Sustained release of antiinfectives

ABSTRACT

Provided are lipid antiinfective formulations substantially free of anionic lipids with a lipid to antiinfective ratio is about 1:1 to about 4:1, and a mean average diameter of less than about 1 μm. Also provided is a method of preparing a lipid antiinfective formulation comprising an infusion process. Also provided are lipid antiinfective formulations wherein the lipid to drug ratio is about 1:1 or less, about 0.75:1 or less, or about 0.50:1 or less prepared by an in line fusion process. The present invention also relates to a method of treating a patient with a pulmonary infection comprising administering to the patient a therapeutically effective amount of a lipid antiinfective formulation of the present invention. The present invention also relates to a method of treating a patient for cystic fibrosis comprising administering to the patient a therapeutically effective amount of a lipid antiinfective formulation of the present invention.

RELATED APPLICATION

This application is a continuation of U.S. patent application Ser. No.12/748,756, filed Mar. 29, 2010, now U.S. Pat. No. 8,802,137, which is acontinuation of U.S. patent application Ser. No. 11/185,448, filed Jul.19, 2005, now U.S. Pat. No. 7,718,189, which is a continuation-in-partof U.S. patent application Ser. No. 11/023,971, filed Dec. 28, 2004, nowabandoned, which is a continuation-in-part of U.S. patent applicationSer. No. 10/696,389, filed Oct. 29, 2003, now U.S. Pat. No. 7,544,369,which claims the benefit of priority from U.S. Provisional PatentApplication Ser. No. 60/421,923, filed Oct. 29, 2002, each of which ishereby incorporated by reference in its entirety.

INTRODUCTION

Certain sustained release technology suitable for administration byinhalation employs liposomes and lipid complexes to provide prolongedtherapeutic effect of drug in the lung and systemically by sustainedrelease and the ability to target and enhance the uptake of drug intosites of disease. The present invention comprises a liposomalantiinfective, and methods for treatment of pulmonary infections usingliposomal or lipid-complexed antiinfective.

As reported in Good an and Gilman's The Pharmaceutical Basis ofTherapeutics, Eighth Edition, “Since the incidence of nephrotoxicity andototoxicity is related to the concentration to which an aminoglycosideaccumulates, it is critical to reduce the maintenance dosage of thesedrugs in patients with impaired renal function.” Since aminoglycosidescan produce vestibular or auditory dysfunction and nephrotoxicityregardless of a patient's impairments, it is important generally toreduce maintenance dosages. The present invention provides dramaticreductions in toxicity thus allowing higher doses than usual.

Cystic fibrosis (CF) patients have thick mucous and/or sputum secretionsin the lungs, frequent consequential infections, and biofilms resultingfrom bacterial colonizations. All these fluids and materials createbarriers to effectively targeting infections with antiinfectives. Thepresent invention overcomes these barriers, and even allows reduceddosing (in amount or frequency), thereby reducing the drug load onpatients. For lung infections generally, the dosing schedule provided bythe invention provides a means of reducing drug load.

For a liposomal drug delivery system, it is often desirable to lower thelipid-to-drug (L/D) ratio as much as possible to minimize the lipid loadto avoid saturation effects in the body. For lung delivery byinhalation, this may be particularly true because for chronic use,dosing of liposomes could outpace clearance thus limiting theadministration and thus effectiveness of the drug product. A lower L/Dratio would allow more drug to be given before the dosing/clearancethreshold is met.

SUMMARY OF INVENTION

Via infusion methods disclosed herein, liposomes substantially free ofanionic lipids of modest size (<1 μm) that entrap antiinfectives at alipid/antiinfective weight ratio of typically about 4:1 to about 0.5:1have been created. The captured volumes of liposomes have been measured,and from these numbers one is able to calculate what the theoreticalentrapment should be if the antiinfective behaved as an ideal solute(i.e., does not interact with the liposome membrane but entraps ideallyalong with water). From this comparison, entrapment numbers that are3-5× higher than expected are observed, indicating that a specialinteraction is occurring that allows greater than expected entrapment,and lower than expected lipid/antiinfective ratios. The solution inwhich the liposomes form contains a concentration of antiinfective, theconcentration of antiinfective inside the liposomes should be about thesame concentration as in the solution. However, the internalantiinfective concentration is calculated to be at least about 3×higher.

In part, the present invention features a liposomal anitiinfectiveformulation comprising a lipid formulation and an antiinfective, whereinthe lipid formulation is substantially free of anionic lipids, andwherein the weight ratio of lipid to antiinfective is about 4:1 to about1:1. In certain embodiments, the weight ratio of lipid to antiinfectiveis about 3:1 to about 1:1, 2:1 to about 1:1, or about 1:1.

In another embodiment, the present invention relates to a lipidformulation comprising an antiinfective wherein the lipid toantiinfective ratio is about 1:1 or less, about 0.75:1 or less, or about0.5:1 or less.

In certain embodiments, the lipid antiinfective formulation comprises aliposome having a mean diameter of about 0.2 μm to about 1.0 μm. Incertain other embodiments, the mean diameter is about 0.2 μm to about0.5 μm. In certain other embodiments, the mean diameter is about 0.2 μmto about 0.3 μm.

In certain embodiments, the antiinfective can be any antiinfectivecommonly known in the art. In certain embodiments, the antiinfective canbe an aminoglycoside including, but not limited to, amikacin,tobramycin, or gentamicin, or a pharmaceutically acceptable saltthereof.

In certain embodiments, the lipid formulation comprises a neutral lipid.In certain embodiments, the lipid formulation is free of anionic lipids.In certain other embodiments, the lipid is a phospholipid, including butnot limited to, a phosphatidyl choline such as dipalmitoylphosphatidylcholine or dioleoylphosphatidyl choline; or the lipid can be a steroidsuch as a sterol, including, but not limited to, cholesterol; or thelipid can be a combination thereof.

In part, the present invention features a method of preparing the lipidantiinfective formulation described above comprising infusing an aqueousor alcoholic solution or mixture of the antiinfective with alipid-alcohol solution or mixture at a temperature below the phasetransition of at least one of the lipid components of the neutral lipid,wherein infusing is done from above. In certain embodiments, the alcoholis ethanol.

In certain embodiments, the concentration of the lipid-alcohol solutionor mixture is about 10 to about 30 mg/mL. In certain embodiments, theconcentration of the antiinfective aqueous or alcoholic solution ormixture is about 20 to about 70 mg/mL. In certain embodiments, theconcentration of the neutral lipid-alcohol solution or mixture is about10 to about 30 mg/mL, and the concentration of the antiinfective aqueousor alcoholic solution or mixture is about 20 to about 70 mg/mL. However,one of ordinary skill in the art will appreciate that concentrations mayvary or otherwise be optimized depending on the lipid and/orantiinfective involved.

In certain embodiments, the present invention relates to theaforementioned lipid formulation, wherein the antiinfective is selectedfrom the following: an aminoglycoside, a tetracycline, a sulfonamide,p-aminobenzoic acid, a diaminopyrimidine, a quinolone, a β-lactam, aβ-lactam and a β-lactamase inhibitor, chloraphenicol, a macrolide,linomycin, clindamycin, spectinomycin, polymyxin B, colistin,vancomycin, bacitracin, isoniazid, rifampin, ethambutol, ethionamide,aminosalicylic acid, cycloserine, capreomycin, a sulfone, clofazimine,thalidomide, a polyene antifungal, flucytosine, imidazole, triazole,griseofulvin, terconazole, butoconazole ciclopirax, ciclopirox olamine,haloprogin, tolnaftate, naftifine, terbinafine, or combination thereof.In certain embodiments, the present invention relates to theaforementioned lipid formulation, wherein the antiinfective is anaminoglycoside. In a further embodiment, the antiinfective is anaminoglycoside selected from the following: amikacin, gentamicin, ortobramycin. In a further embodiment, the antiinfective is amikacin. In afurther embodiment, the antiinfective is gentamicin. In a furtherembodiment, the antiinfective is tobramycin.

In certain embodiments, the present invention relates to theaforementioned lipid formulation, wherein the lipid formulation is aliposome.

In certain embodiments, the present invention relates to theaforementioned lipid formulation, wherein the lipid formulationcomprises a phospholipid. In certain embodiments, the lipid formulationcomprises a steroid. In certain embodiments, the lipid formulationcomprises a sterol. In certain embodiments, the lipid formulationcomprises dipalmitoylphosphatidylcholine (DPPC). In certain embodiments,the lipid formulation comprises cholesterol. In certain embodiments, thelipid formulation comprises a phospholipid and a steroid. In certainembodiments, the lipid formulation comprises a phospholipid and asterol. In certain embodiments, the lipid formulation comprises DPPC andcholesterol. In certain embodiments, the present invention relates tothe aforementioned formulation, wherein the lipid formulation comprisesDPPC, dioleoylphosphatidylcholine (DOPC), and cholesterol.

In certain embodiments, the present invention relates to theaforementioned formulation, wherein the lipid formulation comprises DPPCand cholesterol in a mole ratio of about 20:1, 10:1, 5:1, 2:1, or 1:1.

In certain embodiments, the present invention relates to theaforementioned formulation, wherein the lipid formulation comprisesDPPC, DOPC, and cholesterol in a mole ratio of about 5-20:1-20:0.5-1.

In certain embodiments, the present invention relates to theaforementioned lipid formulation, wherein the lipid formulation is aLiposome and the antiinfective is amikacin.

In certain embodiments, the present invention relates to theaforementioned lipid formulation, wherein the lipid formulation is aliposome, the antiinfective is amikacin, and the lipid formulationcomprises a phospholipid and a sterol.

In certain embodiments, the present invention relates to theaforementioned lipid formulation, wherein the lipid formulation is aliposome, the antiinfective is amikacin, and the lipid formulationcomprises a DPPC and a cholesterol.

In another embodiment, the present invention relates a method ofpreparing a lipid formulation comprising an antiinfective comprising:mixing a stream of a lipid solution or mixture, with a stream of anantiinfective solution or mixture, wherein the two streams are mixed inline. In certain embodiments, the two streams enter a Y-connector priorto mixing in line.

In certain embodiments, the present invention relates to theaforementioned method, wherein the stream of a lipid solution ormixture, and the stream of an antiinfective solution or mixture aremixed at a total flow rate of about 700 to about 900 mL/min. In certainembodiments, the stream of a lipid solution or mixture, and the streamof an antiinfective solution or mixture are mixed at a total flow rateof about 800 mL/min. In certain embodiments, the stream of a lipidsolution or mixture is added at a flow rate of about 200 to about 400mL/min. In certain embodiments, the stream of a lipid solution ormixture is added at a flow rate of about 300 mL/min. In certainembodiments, the stream of an antiinfective solution or mixture is addedat a flow rate of about 400 to about 600 mL/min. In certain embodiments,the stream of an antiinfective solution or mixture is added at a flowrate of about 500 mL/min. In certain embodiments, the stream of a lipidsolution or mixture is added at a flow rate of about 300 mL/min, and thestream of an antiinfective solution or mixture is added at a flow rateof about 500 mL/min.

In certain embodiments, the present invention relates to theaforementioned method, wherein the temperature of the combined streamsis about 30-40° C. In certain embodiments, the temperature of the lipidsolution or mixture is about 30° C., and the temperature of theantiinfective solution or mixture is about 30° C. In certainembodiments, the temperature of the lipid solution or mixture is about50° C., and the temperature of the antiinfective solution or mixture isroom temperature.

In certain embodiments, the present invention relates to theaforementioned method, wherein the method of preparing a lipidformulation comprising an antinfective further comprises the step ofdiluting the combined streams with water at least about 20 seconds aftermixing.

In certain embodiments, the present invention relates to theaforementioned method, wherein the concentration of the antiinfectivesolution or mixture is about 30 to about 50 mg/mL. In certainembodiments, the concentration of the antiinfective solution or mixtureis about 40 to about 50 mg/mL.

In certain embodiments, the present invention relates to theaforementioned method, wherein the stream of a lipid solution or mixtureis added at a flow rate of about 300 mL/min, and the stream of anantiinfective solution or mixture is added at a flow rate of about 500mL/min; the temperature of the combined streams is about 30-40° C.; thecombined streams are diluted with water at least about 20 seconds aftermixing; and the concentration of the antiinfective solution or mixtureis about 40 to about 50 mg/mL.

In certain embodiments, the present invention relates to theaforementioned method, wherein the solutions or mixtures are aqueous oralcoholic. In certain embodiments, the present invention relates to theaforementioned method, wherein the lipid formulation is a liposome.

In certain embodiments, the present invention relates to theaforementioned method, wherein the antiinfective is selected from thefollowing: an aminoglycoside, a tetracycline, a sulfonamide,p-aminobenzoic acid, a diaminopyrimidine, a quinolone, a β-lactam, aβ-lactam and a β-lactamase inhibitor, chloraphenicol, a macrolide,linomycin, clindamycin, spectinomycin, polymyxin B, colistin,vancomycin, bacitracin, isoniazid, rifampin, ethambutol, ethionamide,aminosalicylic acid, cycloserine, capreomycin, a sulfone, clofazimine,thalidomide, a polyene antifungal, flucytosine, imidazole, triazole,griseofulvin, terconazole, butoconazole ciclopirax, ciclopirox olamine,haloprogin, tolnaftate, naftifine, terbinafine, or combination thereof.In certain embodiments, the antiinfective is an aminoglycoside. Incertain embodiments, the antiinfective is an aminoglycoside selectedfrom the following: amikacin, gentamicin, or tobramycin. In certainembodiments, the antiinfective is amikacin. In certain embodiments, theantiinfective is gentamicin. In certain embodiments, the antiinfectiveis tobramycin.

In certain embodiments, the present invention relates to theaforementioned method, wherein the lipid comprises a phospholipid. Incertain embodiments, the lipid comprises a steroid. In certainembodiments, the lipid comprises a sterol. In certain embodiments, thelipid comprises DPPC. In certain embodiments, the lipid comprisescholesterol. In certain embodiments the lipid comprises a phospholipidand a sterol. In certain embodiments, the lipid comprises DPPC andcholesterol.

In certain embodiments, the present invention relates to theaforementioned method, wherein the lipid formulation is a liposome andthe antiinfective is amikacin.

In certain embodiments, the present invention relates to theaforementioned method, wherein the lipid formulation is a liposome, theantiinfective is amikacin, and the lipid comprises a phospholipid and asterol.

In certain embodiments, the present invention relates to theaforementioned method, wherein the lipid formulation is a liposome, theantiinfective is amikacin, and the lipid comprises DPPC and cholesterol.

In certain embodiments, the present invention relates to theaforementioned method, wherein the lipid formulation has a lipid toantiinfective ratio of about 1:1 or less.

In certain embodiments, the present invention relates to theaforementioned method, wherein the lipid formulation has a lipid toantiinfective ratio of about 0.75:1 or less.

In certain embodiments, the present invention relates to theaforementioned method, wherein the lipid formulation has a lipid toantiinfective ratio of about 0.5:1 or less.

In certain embodiments, the present invention relates to theaforementioned method, wherein the lipid formulation is a liposome, theantiinfective is amikacin, the lipid comprises DPPC and cholesterol, andthe lipid to antiinfective ratio is about 1:1 or less.

In another embodiment, the present invention relates to a method oftreating pulmonary infections in a patient in need thereof comprisingadministering to the patient a therapeutically effective amount of aliposomal antiinfective formulation comprising a lipid formulation andan antiinfective, wherein the dosage of antiinfective is about 100mg/day or less. In a further embodiment, the dosage amount ofantiinfective is about 30 mg to about 50 mg every other day. In afurther embodiment, the dosage amount of antiinfective is about 30 mg toabout 50 mg every third day.

In another embodiment, the present invention relates to theaforementioned method of treating, wherein the liposome has a meandiameter of about 0.2 μm to about 1.0 μm. In a further embodiment, theliposome has a mean diameter of about 0.2 μm to about 0.5 μm, or about0.2 μm to about 0.3 μm.

In another embodiment, the present invention relates to theaforementioned method of treating, wherein the pulmonary infection is aresult of cystic fibrosis.

In another embodiment, the present invention relates to theaforementioned method of treating, wherein the weight ratio of lipid toantiinfective is about 4:1 to about 0.5:1, about 3:1 to about 0.5:1,about 2:1 to about 0.5:1, or about 1:1 to about 0.5:1.

In another embodiment, the present invention relates to theaforementioned method of treating, wherein the antiinfective is selectedfrom the following: an aminoglycoside, a tetracycline, a sulfonamide,p-aminobenzoic acid, a diaminopyrimidine, a quinolone, a β-lactam, aβ-lactam and a β-lactamase inhibitor, chloraphenicol, penicillins,cephalosporins, a macrolide, linomycin, clindamycin, coricosteroids,prostaglandin, spectinomycin, polymyxin B, colistin, vancomycin,bacitracin, isoniazid, rifampin, ethambutol, ethionamide, aminosalicylicacid, cycloserine, capreomycin, a sulfone, clofazimine, thalidomide, apolyene antifungal, flucytosine, imidazole, triazole, griseofulvin,terconazole, butoconazole ciclopirax, ciclopirox olamine, haloprogin,tolnaftate, naftifine, terbinafine, or combination thereof. In anotherembodiment, the antiinfective is an aminoglycoside. In anotherembodiment, the antiinfective is amikacin.

In another embodiment, the present invention relates to theaforementioned method of treating, wherein the lipid formulationcomprises neutral lipids. In another embodiment, the lipids that make upthe lipid formulation are all neutral lipids. In another embodiment, theliposome is free of anionic lipids. In another embodiment, the lipidformulation comprises a phospholipid. In another embodiment, the lipidformulation comprises a sterol. In another embodiment, the lipidformulation comprises DPPC and cholesterol.

In another embodiment, the present invention relates to theaforementioned method of treating, wherein the antiinfective isamikacin, and the lipid formulation comprises DPPC and cholesterol.

In another embodiment, the present invention relates to theaforementioned method of treating, wherein the antiinfective isamikacin, the weight ratio of lipid to antiinfective is about 4:1 toabout 1:1, and the lipid formulation comprises DPPC and cholesterol. Ina further embodiment, the weight ratio is about 3:1 to about 1:1, 2:1 toabout 1:1, or about 1:1.

In another embodiment, the present invention relates to theaforementioned method of treating, wherein the antiinfective isamikacin, the weight ratio of lipid to antiinfective is about 4:1 toabout 1:1, the lipid formulation comprises DPPC and cholesterol, and thepulmonary infection is a result of cystic fibrosis. In a furtherembodiment, the weight ratio is about 3:1 to about 1:1, 2:1 to about1:1, or about 1:1.

In another embodiment, the present invention relates to theaforementioned method of treating, wherein the antiinfective isamikacin, the weight ratio of lipid to antiinfective is about 4:1 toabout 0.5:1, the lipid formulation comprises DPPC and cholesterol, andthe liposome has a mean diameter of about 0.1 μm to about 0.5 μm. In afurther embodiment, the mean diameter is about 0.2 μm to about 0.4 μm,or about 0.2 μm to about 0.3 μm.

In another embodiment, the present invention relates to theaforementioned method of treating, wherein the antiinfective isamikacin, the weight ratio of lipid to antiinfective is about 4:1 toabout 0.5:1, the lipid formulation comprises DPPC and cholesterol, thepulmonary infection is the result of cystic fibrosis, and the liposomehas a mean diameter of about 0.1 μm to about 1.0 μm. In a furtherembodiment, the mean diameter is about 0.2 μm to about 0.5 μm, or about0.2 μm to about 0.3 μm.

These embodiments of the present invention, other embodiments, and theirfeatures and characteristics, will be apparent from the description,drawings and claims that follow.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the cross sectional diagram of the sputum/biofilm seen inpatients with cystic fibrosis.

FIG. 2 depicts the graphical representation of the targeting and depoteffect of the drug of the present invention.

FIGS. 3 and 4 depict graphical representations of bacteriology ofamikacin in various forms.

FIG. 5 depicts a graphical representation of sustained release forliposomal/complexed amikacin and tobramycin.

FIG. 6 depicts data on free or complexed ciprofloxacin.

FIG. 7 depicts a graphical representation of drug residence in the lunggiven various dosing schedules.

FIG. 8 depicts graphically the two-stream in-line infusion process ofpreparing liposomal antiinfective formulations.

FIG. 9 depicts miscibility of amikacin sulfate with ethanol/water. Linesrepresent maximal amikacin concentration (base) miscible with ethanolsolution at room temperature (RT) and 40° C. At higher concentrationsamikacin forms a separate liquid phase (coacervates), which laterprecipitates as crystals. Vertical lines show ethanol concentration inthe lipid/amikacin infusion mixture (300/500 parts) and after addingwater 200 parts.

DETAILED DESCRIPTION

The present invention discloses a lipid formulation comprising anantiinfective wherein the size and lipid to drug ratios are smaller thanpreviously known. The present invention also discloses a method ofpreparing these lipid formulations.

1. Definitions

For convenience, before further description of the present invention,certain terms employed in the specification, examples and appendedclaims are collected here. These definitions should be read in light ofthe remainder of the disclosure and understood as by a person of skillin the art. Unless defined otherwise, all technical and scientific termsused herein have the same meaning as commonly understood by a person ofordinary skill in the art.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e. to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

The term “bioavailable” is art-recognized and refers to a form of thesubject invention that allows for it, or a portion of the amountadministered, to be absorbed by, incorporated to, or otherwisephysiologically available to a subject or patient to whom it isadministered.

The terms “comprise” and “comprising” are used in the inclusive, opensense, meaning that additional elements may be included.

The terms “encapsulated” and “encapsulating” are refers to adsorption ofantiinfectives on the surface of lipid based formulation, association ofantiinfectives in the interstitial region of bilayers or between twomonolayers, capture of antiinfectives in the space between two bilayers,or capture of antiinfectives in the space surrounded by the inner mostbilayer or monolayer.

The term “including” is used herein to mean “including but not limitedto”. “Including” and “including but not limited to” are usedinterchangeably.

The term “lipid antiinfective formulation,” or “Lip-antiinfective,” or“Lip-An” discussed herein is any form of antiinfective composition whereat least about 1% by weight of the antiinfective is associated with thelipid either as part of a complex with the lipid, or as a liposome wherethe antibiotic may be in the aqueous phase or the hydrophobic bilayerphase or at the interfacial headgroup region of the liposomal bilayer.Preferably, at least about 5%, or at least about 10%, or at least about20%, or at least about 25%, can be so associated. Association can bemeasured by separation through a filter where lipid and lipid-associatedantiinfective is retained and free antiinfective is in the filtrate. A“liposomal antiinfective formulation” is a lipid antiinfectiveformulation wherein the lipid formulation is the form of a liposome.

The term “mammal” is known in the art, and exemplary mammals includehumans, primates, bovines, porcines, canines, felines, and rodents(e.g., mice and rats).

A “patient,” “subject” or “host” to be treated by the subject method maymean either a human or non-human animal.

The term “pharmaceutically-acceptable salts” is art-recognized andrefers to the relatively non-toxic, inorganic and organic acid additionsalts of compounds, including, for example, those contained incompositions of the present invention.

The term “solvent infusion” is a process that includes dissolving one ormore lipids in a small, preferably minimal, amount of a processcompatible solvent to form a lipid suspension or solution (preferably asolution) and then adding the solution to an aqueous medium containingbioactive agents. Typically a process compatible solvent is one that canbe washed away in a aqueous process such as dialysis. The compositionthat is cool/warm cycled is preferably formed by solvent infusion, withethanol infusion being preferred. Alcohols are preferred as solvents.“Ethanol infusion,” a type of solvent infusion, is a process thatincludes dissolving one or more lipids in a small, preferably minimal,amount of ethanol to form a lipid solution and then adding the solutionto an aqueous medium containing bioactive agents. A “small” amount ofsolvent is an amount compatible with forming liposomes or lipidcomplexes in the infusion process. The term “solvent infusion” may alsoinclude an in-line infusion process where two streams of formulationcomponents are first mixed in-line.

The term “substantially free” is art recognized and refers to a trivialamount or less.

The term “therapeutic agent” is art-recognized and refers to anychemical moiety that is a biologically, physiologically, orpharmacologically active substance that acts locally or systemically ina subject. Examples of therapeutic agents, also referred to as “drugs”,are described in well-known literature references such as the MerckIndex, the Physicians Desk Reference, and The Pharmacological Basis ofTherapeutics, and they include, without limitation, medicaments;vitamins; mineral supplements; substances used for the treatment,prevention, diagnosis, cure or mitigation of a disease or illness;substances which affect the structure or function of the body; orpro-drugs, which become biologically active or more active after theyhave been placed in a physiological environment.

The phrase “therapeutically effective amount” as used herein means thatamount of a compound, material, or composition comprising a lipidantiinfective formulation according to the present invention which iseffective for producing some desired therapeutic effect by inhibitingpulmonary infections.

The term “treating” is art-recognized and refers to curing as well asameliorating at least one symptom of any condition or disease. The term“treating” also refers to prophylactic treating which acts to defendagainst or prevent a condition or disease.

2. Antiinfectives

Antiinfectives are agents that act against infections, such asbacterial, mycobacterial, fungal, viral or protozoal infections.Antiinfectives covered by the invention include but are not limited toaminoglycosides (e.g., streptomycin, gentamicin, tobramycin, amikacin,netilmicin, kanamycin, and the like), tetracyclines (such aschlortetracycline, oxytetracycline, methacycline, doxycycline,minocycline and the like), sulfonamides (e.g., sulfanilamide,sulfadiazine, sulfamethaoxazole, sulfisoxazole, sulfacetamide, and thelike), paraaminobenzoic acid, diaminopyrimidines (such as trimethoprim,often used in conjunction with sulfarnethoxazole, pyrazinamide, and thelike), quinolones (such as nalidixic acid, cinoxacin, ciprofloxacin andnorfloxacin and the like), penicillins (such as penicillin G, penicillinV, ampicillin, amoxicillin, bacampicillin, carbenicillin, carbenicillinindanyl, ticarcillin, azlocillin, mezlocillin, piperacillin, and thelike), penicillinase resistant penicillin (such as methicillin,oxacillin, cloxacillin, dicloxacillin, nafcillin and the like), firstgeneration cephalosporins (such as cefadroxil, cephalexin, cephradine,cephalothin, cephapirin, cefazolin, and the like), second generationcephalosporins (such as cefaclor, cefamandole, cefonicid, cefoxitin,cefotetan, cefuroxime, cefuroxime axetil; cefmetazole, cefprozil,loracarbef, ceforanide, and the like), third generation cephalosporins(such as cefepime, cefoperazone, cefotaxime, ceftizoxime, ceftriaxone,ceftazidime, cefixime, cefpodoxime, ceftibuten, and the like), otherbeta-lactams (such as imipenem, meropenem, aztreonam, clavulanic acid,sulbactam, tazobactam, and the like), betalactamase inhibitors (such asclavulanic acid), chlorampheriicol, macrolides (such as erythromycin,azithromycin, clarithromycin, and the like), lincomycin, clindamycin,spectinomycin, polymyxin B, polymixins (such as polymyxin A, B, C, D, E1(colistin A), or E2, colistin B or C, and the like) colistin,vancomycin, bacitracin, isoniazid, rifampin, ethambutol, ethionamide,aminosalicylic acid, cycloserine, capreomycin, sulfones (such asdapsone, sulfoxone sodium, and the like), clofazimine, thalidomide, orany other antibacterial agent that can be lipid encapsulated.Antiinfectives can include antifungal agents, including polyeneantifungals (such as amphotericin B, nystatin, natamycin, and the like),flucytosine, imidazoles (such as n-ticonazole, clotrimazole, econazole,ketoconazole, and the like), triazoles (such as itraconazole,fluconazole, and the like), griseofulvin, terconazole, butoconazoleciclopirax, ciclopirox olamine, haloprogin, tolnaftate, naftifine,terbinafine, or any other antifungal that can be lipid encapsulated orcomplexed. Discussion and the examples are directed primarily towardamikacin but the scope of the application is not intended to be limitedto this antiinfective. Combinations of drugs can be used.

Particularly preferred antiinfectives include the aminoglycosides, thequinolones, the polyene antifungals and the polymyxins.

Also included as suitable antiinfectives used in the lipid antiinfectiveformulations of the present invention are pharmaceutically acceptableaddition salts and complexes of antiinfectives. In cases wherein thecompounds may have one or more chiral centers, unless specified, thepresent invention comprises each unique racemic compound, as well aseach unique nonracemic compound.

In cases in which the antiinfectives have unsaturated carbon-carbondouble bonds, both the cis (Z) and trans (E) isomers are within thescope of this invention. In cases wherein the antiinfectives may existin tautomeric forms, such as keto-enol tautomers, such as

each tautomeric form is contemplated as being included within thisinvention, whether existing in equilibrium or locked in one form byappropriate substitution with R′. The meaning of any substituent at anyone occurrence is independent of its meaning, or any other substituent'smeaning, at any other occurrence.

Also included as suitable antiinfectives used in the lipid antiinfectiveformulations of the present invention are prodrugs of the platinumcompounds. Prodrugs are considered to be any covalently bonded carrierswhich release the active parent compound in vivo.

3. Pulmonary Infections

Among the pulmonary infections (such as in cystic fibrosis patients)that can be treated with the methods of the invention are Pseudomonas(e.g., P. aeruginosa, P. paucimobilis, P. putida, P. fluorescens, and P.acidovorans), staphylococcal, Methicillinresistant Staphylococcus aureus(MRSA), streptococcal (including by Streptococcus pneumoniae),Escherichia coli, Klebsiella, Enterobacter, Serratia, Haemophilus,Yersinia pesos, Burkholderia pseudomallei, B. cepacia, B. gladioli, B.multivorans, B. vietnarniensis, Mycobacterium tuberculosis, M. aviumcomplex (MAC) (M. avium and M. intracellulare), M. kansasii, M. xenopi,M. marinum, M. ulcerans, or M. fortuitum complex (M. fortuitum and M.chelonei) infections.

4. Methods of Treatment

In one embodiment the present invention comprises a method of treatmentcomprising administration of a therapeutically effective amount of alipid antiinfective formulation.

Where no specific dosage is provided below, the preferred dosage of theinvention is 50% or less, 35% or less, 20% or less, or 10% or less, ofthe minimum free drug (which of course can be a salt) amount that iseffective, if delivered to the lungs via a nebulizer, to reduce the CFUcount in the lungs by one order of magnitude over the course of a 14-daytreatment. The comparative free drug amount is the cumulative amountthat would be used in the dosing period applied with the drugadministration of the invention. The comparative minimum free drugdefined in this paragraph is a “comparative free drug amount.”

The non-CF treating embodiments of the invention can be used with anyanimal, though preferably with humans. Relative amounts in a givenanimal are measured with respect to such animal.

The dosing schedule is preferably once a day or less. In preferredembodiments, the dosing schedule is once every other day, every thirdday, every week, or less. For example, the dosing schedule can be everyother day or less, using 50% or less of the comparative free drugamount. Or, for example, the dosing can be daily using 35% or less ofthe comparative free drug amount. See FIGS. 3 and 4 for animal datashowing that the lipid antiinfective formulations of the presentinvention are more efficacious than the free drug.

To treat infections, the effective amount of the antiinfective will berecognized by clinicians but includes an amount effective to treat,reduce, ameliorate, eliminate or prevent one or more symptoms of thedisease sought to be treated or the condition sought to be avoided ortreated, or to otherwise produce a clinically recognizable change in thepathology of the disease or condition. Amelioration includes reducingthe incidence or severity of infections in animals treatedprophylactically. In certain embodiments, the effective amount is oneeffective to treat or ameliorate after symptoms of lung infection havearisen. In certain other embodiments, the effective amount is oneeffective to treat or ameliorate the average incidence or severity ofinfections in animals treated prophylactically (as measured bystatistical studies).

Liposome or other lipid delivery systems can be administered forinhalation either as a nebulized spray, powder, or aerosol, or byintrathecal administration. Inhalation administrations are preferred.The overall result is a less frequent administration and an enhancedtherapeutic index compared to free drug or parenteral form of the drug.Liposomes or other lipid formulations are particularly advantageous dueto their ability to protect the drug while being compatible with thelung lining or lung surfactant.

The present invention includes methods for treatment of pulmonarygram-negative infections. One usefully treated infection is chronicpseudomonal infection in CF patients. Known treatments of lunginfections (such as in CF patients) with aminoglycoside generallycomprise administering approximately 200-600 mg of amikacin ortobramycin per day via inhalation. The present invention allows fortreatment by administering, in one preferred embodiment, 100 mg or lessof amikacin per day (or normalized to 100 mg per day or less if dosingless frequent). In yet another embodiment, administration of 60 mg orless of amikacin every day is performed. And in still another embodimentadministration of approximately 30 to 50 mg not more than once every 2days is performed. The most preferred embodiment comprisesadministration of approximately 30 to 50 mg every other day or everythird day.

5. Lipids and Liposomes

The lipids used in the compositions of the present invention can besynthetic, semi-synthetic or naturally-occurring lipids, includingphospholipids, tocopherols, steroids, fatty acids, glycoproteins such asalbumin, anionic lipids and cationic lipids. The lipids may be anionic,cationic, or neutral. In one embodiment, the lipid formulation issubstantially free of anionic lipids. In one embodiment, the lipidformulation comprises only neutral lipids. In another embodiment, thelipid formulation is free of anionic lipids. In another embodiment, thelipid is a phospholipid. Phosholipids include egg phosphatidylcholine(EPC), egg phosphatidylglycerol (EPG), egg phosphatidylinositol (EPI),egg phosphatidylserine (EPS), phosphatidylethanolamine (EPE), and eggphosphatidic acid (EPA); the soya counterparts, soy phosphatidylcholine(SPC); SPG, SPS, SPI, SPE, and SPA; the hydrogenated egg and soyacounterparts (e.g., HEPC, HSPC), other phospholipids made up of esterlinkages of fatty acids in the 2 and 3 of glycerol positions containingchains of 12 to 26 carbon atoms and different head groups in the 1position of glycerol that include choline, glycerol, inositol, serine,ethanolamine, as well as the corresponding phosphatidic adds. The chainson these fatty acids can be saturated or unsaturated, and thephospholipid can be made up of fatty acids of different chain lengthsand different degrees of unsaturation. In particular, the compositionsof the formulations can include dipalmitoylphosphatidylcholine (DPPC), amajor constituent of naturally-occurring lung surfactant as well asdioleoylphosphatidylcholine (DOPC). Other examples includedimyristoylphosphatidylcholine (DMPC) anddimyristoylphosphatidylglycerol (DMPG) dipalmitoylphosphatidcholine(DPPC) and dipalmitoylphosphatidylglycerol (DPPG)distearoylphosphatidylcholine (DSPC) and distearoylphosphatidylglycerol(DSPG), dioleylphosphatidylethanolamine (DOPE) and mixed phospholipidslike palmitoylstearoylphosphatidylcholine (PSPC) andpalmitoylstearoylphosphatidylglycerol (PSPG), driacylglycerol,diacylglycerol, seranide, sphingosine, sphingomyelin and single acylatedphospholipids like mono-oleoyl-phosphatidylethanol amine (MOPE).

The lipids used can include ammonium salts of fatty acids, phospholipidsand glycerides, steroids, phosphatidylglycerols (PGs), phosphatidicacids (PAs), phosphotidylcholines (PCs), phosphatidylinositols (PIs) andthe phosphatidylserines (PSs). The fatty acids include fatty acids ofcarbon chain lengths of 12 to 26 carbon atoms that are either saturatedor unsaturated. Some specific examples include: myristylamine,palmitylamine, laurylamine and stearylamine, dilauroylethylphosphocholine (DLEP), dimyristoyl ethylphosphocholine (DMEP),dipalmitoyl ethylphosphocholine (DPEP) and distearoylethylphosphocholine (DSEP),N-(2,3-di-(9(Z)-octadecenyloxy)-prop-1-yl-N,N,N-trimethylammoniumchloride (DOTMA) and 1,2-bis(oleoyloxy)-3-(trimethylammonio)propane(DOTAP). Examples of steroids include cholesterol and ergosterol.Examples of PGs, PAs, PIs, PCs and PSs include DMPG, DPPG, DSPG, DMPA,DPPA, DSPA, DMPI, DPPI, DSPI, DMPS, DPPS and DSPS, DSPC, DPPG, DMPC,DOPC, egg PC.

Liposomes or lipid antiinfective formulations composed ofphosphatidylcholines, such as DPPC, aid in the uptake by the cells inthe lung such as the alveolar macrophages and helps to sustain releaseof the antiinfective agent in the lung (Gonzales-Rothi et al. (1991)).The negatively charged lipids such as the PGs, PAs, PSs and PIs, inaddition to reducing particle aggregation, can play a role in thesustained release characteristics of the inhalation formulation as wellas in the transport of the formulation across the lung (transcytosis)for systemic uptake. The sterol compounds are believed to affect therelease and leakage characteristics of the formulation.

Liposomes are completely closed lipid bilayer membranes containing anentrapped aqueous volume. Liposomes can be unilamellar vesicles(possessing a single membrane bilayer) or multilamellar vesicles(onion-like structures characterized by multiple membrane bilayers, eachseparated from the next by an aqueous layer). The bilayer is composed oftwo lipid monolayers having a hydrophobic “tail” region and ahydrophilic “head” region. The structure of the membrane bilayer is suchthat the hydrophobic (nonpolar) “tails” of the lipid monolayers orienttoward the center of the bilayer while the hydrophilic “heads” orienttowards the aqueous phase. Lipid antiinfective formulations areassociations lipid and the antiinfective agent. This association can becovalent, ionic, electrostatic, noncovalent, or steric. These complexesare non-liposomal and are incapable of entrapping additional watersoluble solutes. Examples of such complexes include lipid complexes ofamphotencin B (lanai et al., Proc. Nat. Acad. Sci., 85:6122 6126, 1988)and cardiolipin complexed with doxorubicin.

A lipid clathrate is a three-dimensional, cage-like structure employingone or more lipids wherein the structure entraps a bioactive agent. Suchclathrates are included in the scope of the present invention.

Proliposomes are formulations that can become liposomes or lipidcomplexes upon corning in contact with an aqueous liquid. Agitation orother mixing can be necessary. Such proliposomes are included in thescope of the present invention.

Liposomes can be produced by a variety of methods (for example, see,Bally, Cullis et al., Biotechnol Adv. 5(1):194, 1987). Bangham'sprocedure (J. Mol. Biol., J Mol. Biol. 13(1):238-52, 1965) producesordinary multilamellar vesicles (MLVs). Lenk et al. (U.S. Pat. Nos.4,522,803, 5,030,453 and 5,169,637), Fountain et al. (U.S. Pat. No.4,588,578) and Cullis et al. (U.S. Pat. No. 4,975,282) disclose methodsfor producing multilamellar liposomes having substantially equalinterlamellar solute distribution in each of their aqueous compartments.Paphadjopoulos et al., U.S. Pat. No. 4,235,871, discloses preparation ofoligolamellar liposomes by reverse phase evaporation.

Unilamellar vesicles can be produced from MLVs by a number oftechniques, for example, the extrusion of Cullis et al. (U.S. Pat. No.5,008,050) and Loughrey et al. (U.S. Pat. No. 5,059,421). Sonication andhomogenization can be used to produce smaller unilamellar liposomes fromlarger liposomes (see, for example, Paphadjopoulos et al., Biochim.Biophys. Acta., 135:624-638, 1967; Deamer, U.S. Pat. No. 4,515,736; andChapman et al., Liposome Technol., 1984, pp. 1-18).

The original liposome preparation of Bangham et al. (J. Mol. Biol.,1965, 13:238-252) involves suspending phospholipids in an organicsolvent which is then evaporated to dryness leaving a phospholipid filmon the reaction vessel. Next, an appropriate amount of aqueous phase isadded, the mixture is allowed to “swell”, and the resulting liposomeswhich consist of multilamellar vesicles (MLVs) are dispersed bymechanical means. This preparation provides the basis for thedevelopment of the small sonicated unilamellar vesicles described byPapahadjopoulos et al. (Biochim. Biophys, Acta., 1967, 135:624-638), andlarge unilamellar vesicles.

Techniques for producing large unilamellar vesicles (LUVs), such as,reverse phase evaporation, infusion procedures, and detergent dilution,can be used to produce liposomes. A review of these and other methodsfor producing liposomes can be found in the text Liposomes, Marc Ostro,ed., Marcel Dekker, Inc., New York, 1983, Chapter 1, the pertinentportions of which are incorporated herein by reference. See also Szoka,Jr. et al., (1980, Ann. Rev. Biophys. Bioeng., 9:467), the pertinentportions of which are also incorporated herein by reference.

Other techniques that are used to prepare vesicles include those thatform reverse-phase evaporation vesicles (REV), Papahadjopoulos et al.,U.S. Pat. No. 4,235,871. Another class of liposomes that can be used arethose characterized as having substantially equal lamellar solutedistribution. This class of liposomes is denominated as stableplurilamellar vesicles (SPLV) as defined in U.S. Pat. No. 4,522,803 toLenk, et al. and includes monophasic vesicles as described in U.S. Pat.No. 4,588,578 to Fountain, et al. and frozen and thawed multilamellarvesicles (FATMLV) as described above.

A variety of sterols and their water soluble derivatives such ascholesterol hemisuccinate have been used to form liposomes; seespecifically Janoff et al., U.S. Pat. No. 4,721,612, issued Jan. 26,1988, entitled “Steroidal Liposomes.” Mayhew et al, described a methodfor reducing the toxicity of antibacterial agents and antiviral agentsby encapsulating them in liposomes comprising alpha-tocopherol andcertain derivatives thereof. Also, a variety of tocopherols and theirwater soluble derivatives have been used to form liposomes, see Janoffet al., U.S. Pat. No. 5,041,278.

6. Methods of Preparation

A process for forming liposomes or lipid antiinfective formulationsinvolves a “solvent infusion” process. This is a process that includesdissolving one or more lipids in a small, preferably minimal, amount ofa process compatible solvent to form a lipid suspension or solution(preferably a solution) and then infusing the solution into an aqueousmedium containing the antiinfective. Typically a process compatiblesolvent is one that can be washed away in a aqueous process such asdialysis or diafiltration. “Ethanol infusion,” a type of solventinfusion, is a process that includes dissolving one or more lipids in asmall, preferably minimal, amount of ethanol to form a lipid solutionand then infusing the solution into an aqueous medium containing theantiinfective. A “small” amount of solvent is an amount compatible withforming liposomes or lipid complexes in the infusion process. Suchprocesses are described in Lee et al., U.S. patent application Ser. No.10/634,144, filed Aug. 4, 2003, Pilkiewicz et al, U.S. patentapplication Ser. No. 10/383,173, filed Mar. 5, 2003, and Boni et al.,U.S. patent application Ser. No. 10/383,004, filed Mar. 5, 2003, whichapplications are hereby incorporated by reference in their entirety.

The step of infusing the lipid-alcohol solution into the aqueous oralcoholic solution or mixture containing the antiinfective can beperformed above or below the surface of the aqueous or alcoholicsolution or mixture containing the antiinfective. Preferably, the stepis performed above the surface of the solution or mixture.

Liposomes can also be prepared by the methods disclosed in copendingU.S. patent application Ser. No. 10/383,004, filed Mar. 5, 2003; Ser.No. 10/634,144, filed Aug. 4, 2003; Ser. No. 10/224,293, filed Aug. 20,2002; and Ser. No. 10/696,389, filed Oct. 29, 2003, the specificationsof which are incorporated herein in their entirety.

Liposome or lipid formulation sizing can be accomplished by a number ofmethods, such as extrusion, sonication and homogenization techniqueswhich are well known, and readily practiced, by ordinarily skilledartisans. Extrusion involves passing liposomes, under pressure, one ormore times through filters having defined pore sizes. The filters aregenerally made of polycarbonate, but the filters may be made of anydurable material which does not interact with the liposomes and which issufficiently strong to allow extrusion under sufficient pressure.Preferred filters include “straight through” filters because theygenerally can withstand the higher pressure of the preferred extrusionprocesses of the present invention. “Tortuous path” filters may also beused. Extrusion can also use asymmetric filters, such as Anopore™filters, which involves extruding liposomes through a branched-pore typealuminum oxide porous filter.

Liposomes or lipid formulations can also be size reduced by sonication,which employs sonic energy to disrupt or shear liposomes, which willspontaneously reform into smaller liposomes. Sonication is conducted byimmersing a glass tube containing the liposome suspension into the sonicepicenter produced in a bath-type sonicator. Alternatively, a probe typesonicator may be used in which the sonic energy is generated byvibration of a titanium probe in direct contact with the liposome.suspension. Homogenization and milling apparatii, such as the CliffordWood homogenizer, Polytron™ or Microfluidizer, can also be used to breakdown larger liposomes or lipid formulations into smaller liposomes orlipid formulations.

The resulting liposomal formulations can be separated into homogeneouspopulations using methods well known in the art; such as tangential flowfiltration. In this procedure, a heterogeneously sized population ofliposomes or lipid formulations is passed through tangential flowfilters, thereby resulting in a liposome population with an upper and/orlower size limit. When two filters of differing sizes, that is, havingdifferent pore diameters, are employed, liposomes smaller than the firstpore diameter pass through the filter. This filtrate can the be subjectto tangential flow filtration through a second filter, having a smallerpore size than the first filter. The retentate of this filter is aliposomal/complexed population having upper and lower size limitsdefined by the pore sizes of the first and second filters, respectively.

Mayer et al. found that the problems associated with efficiententrapment of lipophilic ionizable bioactive agents such asantineoplastic agents, for example, anthracyclines or vinca alkaloids,can be alleviated by employing transmembrane ion gradients. Aside frominducing greater uptake, such transmembrane gradients can also act toincrease antiinfective retention in the liposomal formulation.

Lipid antiinfective formulations have a sustained antiinfective effectand lower toxicity allowing less frequent administration and an enhancedtherapeutic index. In preclinical animal studies and in comparison toinhaled tobramycin (not-liposomal or lipid-based) at the equivalent doselevel, liposomal amikacin was shown to have, during the time periodshortly after administration to over 24 hours later, drug levels in thelung that ranged from two to several hundred times that of tobramycin.Additionally, liposomal amikacin maintained these levels for well over24 hours. In an animal model designed to mimic the pseudomonas infectionseen in CF patients, liposomal amikacin was shown to significantlyeliminate the infection in the animals' lungs when compared to freeaminoglycosides.

Lung surfactant allows for the expansion and compression of the lungsduring breathing. This is accomplished by coating the lung with acombination of lipid and protein. The lipid is presented as a monolayerwith the hydrophobic chains directed outward. The lipid represents 80%of the lung surfactant, the majority of the lipid beingphosphatidylcholine, 50% of which is dipalmitoyl phosphatidylcholine(DPPC) (Veldhuizen et al, 1998). The surfactant proteins (SP) that arepresent function to maintain structure and facilitate both expansion andcompression of the lung surfactant as occurs during breathing. Of these,SP-B and SP-C specifically have lytic behavior and can lyse liposomes(Hagwood et al., 1998; Johansson, 1998). This lytic behavior couldfacilitate the gradual break-up of liposomes. Liposomes can also bedirectly ingested by macrophages through phagocytosis (Couveur et al.,1991; Gonzales-Roth et al, 1991; Swenson et al, 1991). Uptake ofliposomes by alveolar macrophages is another means by which drugs can bedelivered to the diseased site.

The lipids preferably used to form either liposomal or lipidformulations for inhalation are common to the endogenous lipids found inthe lung surfactant. Liposomes are composed of bilayers that entrap thedesired pharmaceutical. These can be configured as multilamellarvesicles of concentric bilayers with the pharmaceutical trapped withineither the lipid of the different layers or the aqueous space betweenthe layers. The present invention utilizes unique processes to createunique liposomal or lipid antiinfective formulations. Both the processesand the product of these processes are part of the present invention.

6.1 In-Line Infusion Method

In one particularly preferred embodiment, the liposomal antiinfectiveformulations of the present invention are prepared by an in-lineinfusion method where a stream of lipid solution is mixed with a streamof antiinfective solution in-line. For example, the two solutions may bemixed in-line inside a mixing tube preceded by a Y-connector as depictedin FIG. 8. In this way, the in-line infusion method differs from theinfusion method described above, where the lipid solution is infused asa stream into a bulk of antiinfective solution. Surprisingly, thisinfusion method results in lower lipid to drug ratios and higherencapsulation efficiencies. The process may be further improved byoptimizing parameters such as flow rate, temperature, antiinfectiveconcentration, and salt addition after infusion step.

6.1.a Effect of Flow Rates

Individual flow rates were varied while keeping the total flow rate at80 mL/min. To do so, two separate pumps were used set at differentpumping rates. The mixed solutions were infused for 10 s into a beakercontaining NaCl solution such that the final NaCl concentration was 1.5%and the final ethanol concentration did not exceed 30%. After mixing, a1 mL aliquot was run though a Sephadex G-75 gel filtration column toseparate free amikacin from encapsulated. A 1 mL fraction with highestdensity (determined by visual turbidity) was collected for furtheranalysis. The results are presented in Table 1. Increasing thelipid/amikacin flow rate ratio resulted in an almost constant L/D until300/500 mL/min. With further increase of lipid rate, L/D started toincrease and particle size also started getting larger. At the sametime, higher lipid flow rates gave better amikacin recovery(encapsulation efficiency) as more lipid mass was added.

TABLE 1 Effect of flow rates on amikacin encapsulation.* Flow rates AMKAMK AMK mL/min total free Lipid VOL Recovery Batch AMK Lipid mg/mL %mg/mL L/D Size % 1 600 200 1.38 5.3 1.25 0.91 289 14.7 2 550 250 1.805.1 1.90 1.06 305 17.2 3 500 300 2.18 5.2 2.29 1.05 314 22.8 4 450 3501.27 5.8 1.47 1.16 388 26.8 5 400 400 1.05 6.1 1.69 1.61 471 24.9 *Lipidand amikacin solutions were kept at 40° C. Amikacin stock solution was50 mg/mL. NaCl 10% solution was added before infusion to obtain final1.5%. Infusion time was set at 10 s. Mixing tube 10 cm; 6-elementin-line mixer positioned at 0 cm.

Batch 3 with the lipid/amikacin flow rates of 300/500 mL/min showed thebest L/D and particle size, combined with reasonably high amikacinrecovery. Thus it was decided to use these flow rates for all furtherexperiments.

In order to reproduce the results at chosen conditions a fully washedbatch (batch 6) using diafiltration was prepared as presented in Table2. NaCl 10% solution was added into the beaker prior to infusion to makethe final concentration 2% (as compared to 1.5% in the batches in Table1). The resulting L/D (1.71) was not as good as in batch 3 in Table 1and the particle size was higher. This may be due to an adverse effectof high NaCl concentration contacting liposomes in the early stages ofliposome formation. Samples separated (washed) using gel-filtrationcolumns tend to have better L/D than ones washed by diafiltration. Thismay have to do with the different degree of stress liposomes experience,or simply samples separated on the gel filtration column contained afraction of liposomes with better L/D which does not represent the wholepopulation.

TABLE 2 Summary of the fully washed batches. Process parameters variedwere: temperatures, amikacin stock concentration, and other (see Table 3below). All batches were concentrated to nearly a maximum extent, untilthe inlet pressure reached 10 PSI. Temp, C. AMK stock AMK total AMK freeLipid Size VOL Size Batch L/AMK/W mg/mL mg/mL % mg/mL L/D nm SD % 640/40/30 50 36.1 2.7 61.8 1.71 392 43.4 8 50/RT/30 50 48.5 9.6 49.3 1.02332 32.0 9 50/RT/30 50 41.6 5.1 43.2 1.04 359 34.4 10 50/RT/30 50 53.110.2 34.4 0.65 350 28.6 11 50/RT/30 40 20.7 4.8 46.9 2.27 407 35.9 1250/RT/30 40 81.0 1.9 49.4 0.61 341 33.0 13 50/RT/30 30 68.6 1.7 62.50.91 311 22.4 14 50/RT/30 40 79.6 1.6 47.8 0.60 346 37.2 15 50/RT/30 4071.3 2.0 42.3 0.59 353 33.4 16 30/30/30 40 61.9 6.1 51.5 0.83 369 28.417 30/30/30 40 73.8 2.4 57.2 0.77 362 32.6 18 30/30/30 40 74.4 2.3 54.00.73 549 61.7 *The 3^(rd) column represents the temperature of the Lipidand Amikacin solutions just before infusion, and the temperature duringwashing (diafiltration). RT = room temperature. “VOL size” is the volumeof weighted particle size.

TABLE 3 Processing conditions for batches 1-18.* Mixing Mixer NaCl addedtube position Volume Timing to Washing conditions Batch cm cm Stock %parts infusion NaCl % 1st wash 1-5 10 0 VAR VAR before 1.5 (Seph column)6 10 0 10 200 before 1.5 diafiltration 7 10 5 10 100 before 1.5 (Sephcolumn) 8 10 5 10 150 during 1.5 diafiltration 9 10 5 10 150 during 1.5diafiltration 10 10 5 10 100 5′ after 1.5 2 × dilution 11 10 5 10 150imm after 1.5 2 × dilution 12 10 5 H2O 180 20″ after 1.5 2 × dilution 1310 5 H2O 180 30″ after 1.5 2 × dilution 14 10 5 H2O 180 30″ after 1.5diafiltration 15 10 5 1.5 180 30″ after 1.5 diafiltration 16 60 NO 0.9180 during 0.9 diafiltration 17 60 NO 1.5 180 during 1.5 diafiltration18 60 0 1.5 180 during 1.5 diafiltration *Lipid and amikacin solutionswere infused at rates 300/500 mL/min for 30 s (examples 6-10) or 20 s(examples 11-18). Additional aqueous solution (NaCl or water) was added(as parts relative to 500 parts amikacin volume).

6.1.b Effects of Process Temperature.

The settings were kept the same as in batch 3 except that the amount ofNaCl solution added was less, making the final concentration 1.0%.Solution was added again before infusion was initiated because with theshort infusion time it was difficult to make the addition duringinfusion. Also, during infusion the in-line mixer shifted to the end ofthe mixing tube under the pressure of the flow. The position of themixer was 5 cm from the front end of the tube instead of 0 cm for batch3. This may be important, as the L/D ratio obtained at the sametemperature 40/40° C. condition in batch 20 was 0.55, almost half ofthat in batch 3. On comparing amikacin encapsulation at differentinfusion temperatures, one can see that, surprisingly, lowertemperatures gave better L/D. Of the temperatures tested, lipid/amikacintemperatures 30/30° C. and 50/RT gave similar L/D ratios of 0.32 and0.37. Again, as in batches 1-5, the numbers from these washed samples bygel-filtration were low, perhaps less than that if the batches had beenwashed by diafiltration.

TABLE 4 Effect of temperature on amikacin encapsulation.* Temperature,C. AMK AMK VOL mL/min total free Lipid Size Batch Lipid AMK mg/mL %mg/mL L/D nm 19 30 30 4.88 2.8 1.54 0.32 278 20 40 40 3.62 1.5 1.98 0.55335 21 50 50 3.50 1.8 2.74 0.78 309 22 50 RT 5.27 2.9 1.93 0.37 342*Lipid and amikacin solutions were infused at rates 300/500 mL/min for10 s. Amikacin stock solution was 50 mg/mL. NaCl 10% solution was addedbefore infusion to obtain a final 1.0% concentration. Mixing tube 10 cm,6-element in-line mixer positioned at 5 cm.

In separate experiments it was found that mixing of 90% ethanol andwater at either 30° C. and 30° C. or 50° C. and 22° C., respectively,resulted in a similar final temperature of nearly 36° C. This suggeststhat the temperature of the final mixture rather than that of theindividual components is important for amikacin encapsulation. Thetemperatures 50° C./RT were used in examples 6-15. In examples 16-18temperatures of 30° C. and 30° C. for the two streams were used withcomparable results, although a little less amikacin encapsulation wasobserved.

6.1.c. Effect of Post-Infusion Addition of Aqueous Volume

Attention was next focused on the steps of NaCl solution addition andthe washing process. Process parameters were varied in variousdirections. Right after the infusion step at flow rates 300/500, ethanolconcentration in the mixture reaches 34%. Amikacin has limitedsolubility at this concentration (see FIG. 9).

If one starts with 50 mg/mL amikacin stock, then after mixing with thelipid solution there will be more than 30 mg/mL total amikacin where atleast half (15 mg/mL) is free amikacin, assuming 50% encapsulationefficiency. This is higher than the solubility limit at 34% ethanol. Onepossible solution to this problem is to add more water to the vesselwith the lipid/amikacin mixture, thus reducing both ethanol and amikacinconcentration. For example, adding 200 parts of water (or NaCl solution)to 800 parts of lipid/amikacin would reduce ethanol to 27% (FIG. 9).This makes amikacin soluble at 15 mg/mL or even higher depending ontemperature.

In addition, adding NaCl may stabilize osmotic conditions. Whenliposomes are formed and amikacin is encapsulated at an internalconcentration of 200-300 mg/mL, there is only ˜15 mg/mL or so ofamikacin not encapsulated. In the absence of saline this would create anosmotic imbalance, which in turn might lead to leakage of amikacin.Adding 150 parts of 10% NaCl to 800 parts of lipid/amikacin will resultin about 1.5% NaCl final concentration (outside liposomes).

A number of batches were generated where different amounts of NaClsolution (or water in some batches) were added at different timesrelative to the infusion event (see Table 5, compiled from Tables 2 and3). From the table a general trend can be seen, leading to the followingconclusions:

-   -   Some time interval between infusion and addition of the aqueous        volume is required to obtain lower L/D (if a short mixing tube        is used). Of batches 6-15, those with an interval 20 s or longer        had lower L/D. One possible explanation is that liposomes are        not completely formed immediately after mixing of the streams.        When a longer mixing tube is used (batches 16-18), which allows        for a longer mixing time, the time interval is not required.    -   Adding a high concentration NaCl solution to balance osmolality        does not actually help retain amikacin. In fact, adding pure        water at an appropriate time interval resulted in the lowest L/D        and total amikacin concentration.    -   Adding 100 parts NaCl 10% (batch 9) 5 min after infusion gave a        competitive L/D ratio but did not give as good a total amikacin        concentration. It may be that NaCl, when present at early stages        with relatively high ethanol concentrations, leads to increased        aggregation and viscosity.

TABLE 5 Role of aqueous volume and NaCl concentration added to thelipid/amikacin mixture to adjust ethanol concentration. Not all thevariables shown; see Tables 2 and 3. AMK NaCl added AMK Size stock StockVolume Timing to total VOL Batch mg/mL % parts infusion mg/mL L/D nm 650 10 200 before 36.1 1.71 392 8 50 10 150 during 48.5 1.02 332 9 50 10150 during 41.6 1.04 359 10 50 10 100 5′ after 53.1 0.65 350 11 40 10150 imm after 20.7 2.27 407 12 40 H₂O 180 20″ after 81.0 0.61 341 13 30H₂O 180 30″ after 68.6 0.91 311 14 40 H₂O 180 30″ after 79.6 0.60 346 1540 1.5 180 30″ after 71.3 0.59 353 16 40 0.9 180 during 61.9 0.83 369 1740 1.5 180 during 73.8 0.77 362 18 40 1.5 180 during 74.4 0.73 549

6.1.d. Effect of Antiinfective Stock Solution

Previously it was found that using 50 mg/mL amikacin stock solutionproduced the best entrapment. Reducing the amikacin stock concentrationto 40 mg/mL increased L/D when used in conventional processes. With thetwo-stream in-line infusion process, ethanol concentration reacheshigher levels, so the current 50 mg/mL amikacin may not be the optimalconcentration.

Table 6 summarizes the effect of using various amikacin stockconcentrations. 40 mg/mL delivered comparable or better L/D values, andeven improved amikacin recovery. Using less amikacin relative to aconstant amount of lipid, and providing a similar L/D, resulted in ahigher percent encapsulation (batch 12). Further decrease of amikacinstock concentration to 30 mg/mL resulted in a slightly increased L/D,although recovery was still impressive (batch 13).

TABLE 6 Amikacin stock concentration can be reduced while improvingefficiency. Amikacin recovery is calculated based on L/D obtained andassumed 100% lipid recovery. AMK AMK AMK Size AMK stock total free LipidVOL Recovery Batch mg/mL mg/mL % mg/mL L/D nm % 10 50 53.1 10.2 34.40.65 350 37.0 12 40 81.0 1.9 49.4 0.61 341 51.2 13 30 68.6 1.7 62.5 0.91311 45.7 14 40 79.6 1.6 47.8 0.60 346 52.0

Reducing amikacin stock concentration has another implication. itreduces the concentration of free. amikacin in a post-infusionlipid/amikacin mixture, allowing it to remain soluble at higher ethanolconcentration. Assuming that lipid and amikacin are mixed at 300/500ratio, amikacin stock is 50 mg/mL, and encapsulation efficiency is 37%,then initial free amikacin would be ˜20 mg/mL. Similarly, 40 mg/mLamikacin stock with 52% encapsulation would result in ˜12 mg/mL freeamikacin. 30 mg/mL amikacin stock with 46% encapsulation would result in˜10 mg/mL free amikacin.

7. Lipid to Drug Ratio

There are several ways to increase the entrapment of antiinfectives(e.g. aminoglycosides such as amikacin, tobramycin, gentamicin) inliposomes. One way is to make very large liposomes (>1 μm) where theentrapped volume per amount of lipid is large. This approach to achievea smaller L/D ratio is not practical for inhalation (nebulization) ofliposomes because 1) shear stress during nebulization tends to ruptureliposomes in a size dependent manner where larger liposomes (>0.5 μm)suffer greater release and 2) the smaller droplet sizes necessary forgood lung deposition are themselves less than about ˜3 μm. So forinhalation, it is desirable to keep the liposome size as small aspossible to avoid too much release. Currently, the mean diameter for theliposomes disclosed herein is less than about 0.4 μm (see Table 4).

Another approach to decrease L/D is to use negatively charged lipids.The aminoglycosides listed above are highly positively charged with 4 to5 amines per compound. Usually sulfate salts of these aminoglycosidesare used in therapeutic formulations. Along with the multi-cationiccharacter comes strong binding to negatively charged liposomes. Thisresults in greater entrapment during liposome formation. The purpose ofantiinfective formulations is to provide sustained release to the lungenvironment. Rapid clearance of the liposomes by macrophage uptake wouldrun counter to this. It has been well documented that negatively chargedliposomes experience a much higher degree of uptake by macrophages thanneutral liposomes. Therefore, it is desirable to use neutral liposomes.

One group of technologies that allow very high drug entrapment intosmall liposomes is based on gradient loading where a pH gradient,ammonium sulfate gradient, or Mg-sulfate gradient are used to loadamine-drugs into liposomes: see U.S. Pat. Nos. 5,578,320 5,736,1555,837,279 5,922,350 (pH gradient); U.S. Pat. Nos. 5,837,282 5,785,987(Mg-sulfate gradient); and U.S. Pat. No. 5,316,771 (ammonium sulfategradient). These techniques only work for membrane permeable amines(mono-amines where neutral form is permeable like doxorubicin anddaunorubicin). Gradient loading will not work for the certainantiinfectives such as aminoglycosides as they are impermeable (toolarge and too highly charged).

All processes described herein can be easily adapted for large scale,aseptic manufacture. The final liposome size can be adjusted bymodifying the lipid composition, concentration, excipients, andprocessing parameters.

The lipid to drug ratio using the processes of the present invention isabout 4:1 to about 1:1. In another embodiment, the lipid to drug ratiois about 3:1 to about 1:1, 2:1 to about 1:1, about 1:1 or less, about0.75:1 or less, or about 0.5:1 or less. Further the percentage of freeantiinfective, present after the product is dialyzed for a particularduration, is decreased.

8. Results

8.1. Biofilm Barriers of Pulmonary Infections

An obstacle to treating infectious diseases such as Pseudomonasaeruginosa, the leading cause of chronic illness in cystic fibrosispatients is drug penetration within the sputum/biofilm barrier onepithelial cells (FIG. 1). In FIG. 1, the donut shapes represent aliposomal antiinfective formulation, the “+” symbol represents freeantiinfective, the “−” symbol mucin, alginate and DNA, and the solid barsymbol represents Pseudomonas aeruginosa. This barrier is composed ofboth colonized and planktonic P. aeruginosa embedded in alginate orexopolysaccharides from bacteria, as well as DNA from damagedleukocytes, and mucin from lung epithelial cells, all possessing a netnegative charge (Costerton, et al., 1999). This negative charge binds upand prevents penetration of positively charged drugs such asaminoglycosides, rendering them biologically ineffective (Mendelman etal., 1985). Entrapment of antiinfectives within liposomal or lipidformulations could shield or partially shield the antiinfectives fromnon-specific binding to the sputum/biofilm, allowing for liposomal orlipid formulations (with entrapped aminoglycoside) to penetrate (FIG.1).

Amikacin has been shown to have a high degree of resistance to bacterialenzymes, thus providing a greater percent of susceptible clinicalisolates than found for other aminoglycosides including tobramycin andgentamicin (Price et al., 1976). In particular, P. aeruginosa isolatesare far more sensitive to amikacin than other aminoglycosides whileexhibiting no cross-resistance (Damaso et al., 1976).

The sustained release and depot effect of liposomal formulations ofamikacin is clearly seen in FIG. 2. In this study rats were giventobramycin via intratracheal and intravenous administration. The ratswere also given liposomal formulations of amikacin intratracheally atthe same dose (4 mg/rat). The data show that it is only with theliposomal formulation of amikacin that a sustained release and depoteffect is achieved. In fact, 24 hours after dosing, only liposomalformulations of amikacin show significant levels of the drug in theanimal's lungs, while both tobramycin formulations revealed negligiblelevels, primarily due, it is believed to rapid systemic absorption. Thisgreater than a hundred-fold increase of aminoglycoside in the lung forliposomal antiinfective formulations supports the idea of a sustainedrelease liposomal formulation antiinfective that can be takensignificantly less often than the currently approved TOBI® formulation(a tobramycin inhalation solution made by the Chiron Corporation,Ameryville, Calif.).

Moreover, the presence of a sputum/biofilm prevents the penetration ofthe free aminoglycosides due to binding of the antiinfectives to itssurface (FIG. 1). Therefore, doses in excess of 1,000 gm oftobramycin/gram of lung tissue are needed to show a therapeutic effectin CF patients. This is overcome with liposomal formulations ofamikacin. Thus, the therapeutic level of drug is maintained for a longerperiod of time in the liposomal formulations of amikacin compared tofree tobramycin. This facilitation of binding and penetration could alsobe a means by which liposomal formulations of amikacin couldsignificantly reduce bacterial resistance commonly seen to develop whenantibacterials are present in vivo at levels below the minimuminhibitory concentration.

8.2. Pharmacokinetics

The pharmacokinetics of amikacin was determined in rats followingintratracheal (IT) administration of either free tobramycin or liposomalformulations of amikacin. These data were compared to the distributionobtained in the lungs following a tail vein injection of freetobramycin. In all cases a dose of 4 mg/rat was administered. As can beseen in FIG. 2, a much larger deposition of aminoglycoside can bedelivered by IT compared to injection. The depot effect of liposomalantiinfective technology is also demonstrated in that in comparison totobramycin given either IT or IV, a greater than a hundred-fold increasein drug for liposomal formulations of amikacin still remains in thelungs twenty-four hours following administration. Thus, the therapeuticlevel of drug is maintained for a longer period of time in the liposomalformulations of amikacin compared to free tobramycin.

The binding of aminoglycosides to sputum of CF patients is a concern,particularly if this binding reduces the bioactivity of theantiinfective (Hunt et al., 1995). To determine whether liposomalformulations of amikacin can retain biological activity over a prolongedperiod of time, normal rats were administered liposomal formulations ofamikacin by intratracheal instillation. This was followed by its removalat 2 or 24 hours via a bronchial alveolar lavage (BAL) to determinebiological activity. Samples were concentrated by ultrafiltrationfollowed by filtration (0.2 micron) to remove contaminating lungmicrobes. Amikacin concentration was determined employing a TDXinstrument and biological activity determined using a Mueller Hintonbroth dilution assay (Pseudomonas aeruginosa). The results are shown inTable 7.

TABLE 7 Results showing that liposomal formulations of amikacin retainbiological activity over a prolonged period of time. time amikacin inBAL amikacin in filtrate MIC (hours) (μg/mL) (μg/mL) (μg/mL) 2 160 1191.9 24 73 32 4.0

As shown by the above table, the recovered filtered liposomalformulation of amikacin was capable of killing P. aeruginosa in aMueller Hinton broth assay even after 24 hours with an MIC of 4. At 2hours an MIC of 2 was obtained, which is similar to that obtained forthe filtered liposomal/complexed amikacin stock. Thus, the liposomalformulation of amikacin was still active following 24 hours in the lung.At 24 hours free tobramycin at the same dose was undetectable in a BAL.This indicates that not only is the liposomal antiinfective formulationretained in the lung, but it is also freely available to penetrate asputum/biofilm over time. These data combined with the facts as evidentin FIG. 2 and Table 9 (below), that liposomal formulations of amikacinrelease the free antiinfective over time while maintaining high levelsof the antiinfective in the lungs, supports the rationale that thissystem may yield a sustained antiinfective effect over time. This effectshould prove significant in reducing both the bio-burden of thePseudomonas and the development of resistance due to trough levels ofantiinfective.

As an in vitro demonstration of slow release of liposomal formulation ofamikacin and its sustained antiinfective effect, the formulation wasincubated in sputum from patients with Chronic Obstructive PulmonaryDisease (COPD) containing PAOI mucoid Pseudomonas. The liposomalformulation of amikacin was also incubated in alginate containing PAOImucoid Pseudomonas. In both cases sustained and enhanced killing of thePseudomonas over time was observed, as shown in Table 8.

TABLE 8 In vitro killing of Pseudomonas over time. In VitroSputum/Alginate Assay (% survival of PA01 Mucoid Pseudomonas) AmikacinIncubation time at 37° C. conc. 1 h 3 h 6 h 24 (μg/mL) Lip-An-15 Sputum81 15 22 <1 8 Lip-An-15 Alginate 100 59 1 <1 10Classical kill curves are not applicable for liposomal antiinfectiveformulation technology because the liposomal formulations exhibit a slowrelease of antiinfective with an enhanced antiinfective effect. Theliposomal formulation protects the amikacin from the sputum and/oralginate until its release. In time, complete killing is observed,consistent with slow release sustained antiinfective effect model withno interference or inactivation of antiinfective.

The efficacy of liposomal amikacin formulations was studied using amodel for chronic pulmonary infection (Cash et al., 1979) where P.aeruginosa, embedded in an agarose bead matrix, was instilled in thetrachea of rats. This mucoid Pseudomonas animal model was developed toresemble the Pseudomonas infections seen in CF patients. Some of theclinical correlates to CF include: a similar lung pathology; thedevelopment of immune complex disorders; and a conversion to the mucoidphenotype by P. aeruginosa strains (Cantin and Woods, 1999). Rat lungswere infected with over 10⁷ CFUs of a mucoid Pseudomonas (strain PAO1)taken from a CF patient isolate, and subsequently treated with (a) freeaminoglycoside, (b) the lipid vehicle alone as non-drug control, and (c)liposomal amikacin formulation. In addition, formulations were firstscreened on the ability to kill in vitro P. aeruginosa on modifiedKirby-Bauer plates.

Various liposomal amikacin formulations were tested based on eitherdifferent lipid compositions or manufacturing parameters resulting indifferent killing zones in in vitro experiments. This experiment wasdesigned to determine the increase in efficacy obtained with liposomalaminoglycoside formulations over free aminoglycoside. Blank controllipid compositions, two different liposomal amikacin formulations andfree amikacin and free Tobramycin at the same aminoglycosideconcentrations as the liposomal antiinfective formulations werecompared. In addition, a 10 fold higher dose of free amikacin and a 10fold higher dose of free tobramycin were also given. Dosing was IT dailyover seven days. Results (FIG. 3) indicate that liposomal amikacin inthe two formulations (differing in lipid composition) revealed asignificant reduction in CFU levels and were better at reducing CFUsthan free amikacin or free tobramycin at 10-fold higher-dosages. In FIG.3, Lip-An-14 is DPPC/Chol/DOPC/DOPG (42:45:4:9) and 10 mg/mL amikacin,Lip-An-15 is DDPC/Chol (1:1) also at 10 mg/mL. All lipid-lipid andlipid-drug ratios herein are weight to weight.

The next experiment (FIG. 4) was designed to demonstrate the slowrelease and sustained antiinfective capabilities of liposomal amikacinformulations. The dosing was every other day for 14 days, as opposed toevery day for seven days as in the previous experiments. Resultsindicate that liposomal amikacin in the two formulations (differing inlipid composition) had a 10 to 100 times more potent (greater ability toreduce CFU levels) than free amikacin or free tobramycin. A daily humandose of 600 mg TOBI® (a tobramycin inhalation solution made by theChiron Corporation, Ameryville, Calif.), or about 375 mg/m², correspondsto a daily rat dose of 9.4 mg. Thus the data can be directly correlatedto a 10 to 100 fold improvement in human efficacy. It should be notedthat a two-log reduction is the best that can be observed in this model.A 100-fold reduction in P. aeruginosa in sputum assays has beencorrelated with improved pulmonary function (Ramsey et al., 1993). Thesustained release of the liposomal amikacin formulations indicate that alower dose and/or less frequent dosing can be employed to obtain agreater reduction in bacterial growth than can be obtained with freeaminoglycoside.

The efficacy of liposomal amikacin formulation was studied in a modelfor chronic pulmonary infection where P. aeruginosa was embedded in anagarose bead matrix that was instilled via the trachea of Sprague/Dawleyrats. Three days later free amikacin or liposomal amikacin was dosedevery day (FIG. 3) or every other day (FIG. 4) at 1 mg/rat or 10 mg/ratof the given aminoglycoside or 1 mg/rat liposomal amikacin, as well aswith blank liposomes (lipid vehicle) as the control, with five rats pergroup.

The homogenized rat lungs (frozen) following the 14 day experiment wereanalyzed for aminoglycoside content and activity. The clinical chemicalassay was performed using a TDX instrument while the bioassay wasperformed by measuring inhibition zones on agar plates embedded withBacillus subtilis. The results are shown in Table 9:

TABLE 9 Results from liposomal amikacin formulation treated rat lungsinfected with P. aeruginosa. Bioassay Clinical Assay Formulation(microgram/mL) (microgram/mL) Lip-An-14 (1 mg/rat) 9.5 9.1 Lip-An-15 (1mg/rat) 21.5 18.4 Free amikacin (10 mg/rat) nd 2.0 Free tobramycin (10mg/rat) nd 1.4Drug weights are for the drug normalized to the absence of any saltform.

The Table 10 results indicate that aminoglycoside is present and activefor both liposomal antiinfective formulations, while little can bedetected for the free aminoglycoside even at the 10-fold higher dose.These further results establish the sustained release characteristics ofliposomal antiinfective formulations, and also confirm that thatantiinfective which remains is still active. Of the above formulationsonly the free tobramycin (0.1 microgram/mL) exhibited any detectablelevels of aminoglycoside in the kidneys.

The sustained release and depot effect of liposomal amikacin formulationis further demonstrated in FIG. 5. Rats were given a chronic pulmonaryinfection where P. aeruginosa was embedded in an agarose bead matrixthat was instilled via the trachea, using the same beads employed in theefficacy studies. The rats were then given free tobramycin or liposomalamikacin (formulation Lip-An-14) via intratracheal administration at thesame dose (2 mg/rat). The data, measured in microgram antiinfective pergram lung tissue over time, show that liposomal antiinfective exhibits asustained release and depot effect while free tobramycin revealednegligible levels in the lungs by 24 hours, primarily due it is believedto rapid systemic absorption. This greater than a hundred-fold increaseof antiinfective in the lung for liposomal amikacin formulations in aninfected rat supports the idea of a sustained release liposomalantiinfective that can be taken significantly less often than thecurrently approved TOBI® formulation (a tobramycin inhalation solutionmade by the Chiron Corporation, Ameryville, Calif.).

The pharmacokinetics of amikacin was determined in rats followingintratracheal (IT) administration of either free tobramycin or liposomalamikacin. A dose of 2 mg/rat was administered. The depot effect ofliposomal antiinfective technology is demonstrated in that in comparisonto free tobramycin given IT, a greater than a hundred-fold increase indrug for liposomal amikacin still remains in the infected lungstwenty-four hours following administration. Thus, the therapeutic levelof drug is maintained for a longer period of time in the liposomalformulations compared to free tobramycin.

FIG. 7 shows remarkable residence time and accumulation of effectiveamounts of antiinfective in the lungs, a result that establishes thatrelatively infrequent dosings can be used. Each dose is 4 hr. byinhalation (in rat, 3 rats per group, as above) of nebulized liposomalamikacin formulations (DPPC/Chol., 1:1) at 15 mg/mL amikacin. Dosing wasat either day one; day one, three and five; or day one, two, three, fourand five. Rats providing a given data bar were sacrificed after therespective dosing of the data bar. The formulation is made as in theExample.

Similar anti-infectives can be utilized for the treatment ofintracellular infections like pulmonary anthrax and tularemia. Inpulmonary anthrax the anthrax spores reach the alveoli in an aerosol.The inhaled spores are ingested by pulmonary macrophages in the alveoliand carried to the regional tracheobronchial lymph nodes or mediastinallymph nodes via the lymphatics (Pile et al., 1998; Geiser et al., 1968).The macrophage is central in the both the infective pathway and is themajor contributor of host self-destruction in systemic (inhalation)anthrax. In addition to its attributes of sustained release andtargeting, liposomal antiinfective formulation technology can enhancecellular uptake and can use alveolar macrophages and lung epithelialcells in drug targeting and delivery. The possession of thesecharacteristics is believed to facilitate the treatment of theseintracellular infections, which infections occur in the lungs and aretransported by macrophages. More importantly, these characteristicsshould make the antiinfective more effective in that the liposomalantiinfective should be phagocytized by the very cells containing thedisease. The antiinfective would be released intracellularly in atargeted manner, thereby attacking the infection before it isdisseminated. The encapsulated drug can be an already approvedpharmaceutical like ciprofloxacin, tetracycline, erthyromycin oramikacin. Liposomal ciprofloxacin formulations have been developed.

In a study, this compound was administered to mice and compared to bothfree ciprofloxacin administered intratracheally and free ciprofloxacinadministered orally, with all three compounds given at the same dose(FIG. 6). The dose for each mouse was 15 mg/kg, with three mice pergroup. Liposomal ciprofloxacin was in DPPC/Cholesterol (9:1), at 3 mg/mLciprofloxacin, with the formulation produced as in the Example. Thelipid to drug ratio was 12.5:1 by weight. In comparison to orallyadministered ciprofloxacin, liposomal ciprofloxacin was present in themice lungs at amounts over two orders of magnitude higher than freeciprofloxacin. Moreover, only liposomal ciprofloxacin showed levels ofdrug in the lung after 24 hours, while the orally administered drug wasundetectable in less than two hours. This data supports the use ofliposomal ciprofloxacin formulations and other antiinfectives likeaminoglycosides, tetracyclines and macrolides for the treatment and forthe prophylactic prevention of intracellular diseases used bybioterrorists.

8.3. Liposome Parameters

The lipids to be employed are dissolved in ethanol to form alipid-ethanol solution. The lipid-ethanol solution is infused in anaqueous or ethanolic solution containing the molecule of the bioactiveagent to be entrapped. The lipids spontaneously form vesicles.

Table 10 discloses liposomal antiinfective formulation parameters wherethe lipids are DPPC and cholesterol.

TABLE 10 Additional liposomal antiinfective formulation parameters.Amikacin Liposomes (DPPC/Chol) Liposome [Total [Total % of Total MeanAmikacin] Lipid]* Amikacin that L/D Diameter Batch # mg/mL mg/mL isEntrapped (w/w)** (μm) 1 14.7 44.8 96.7 3.2 — 2 21.4 71.3 98.1 3.4 0.363 18.5 46.6 90.2 2.8 0.27 4 9.4 40.6 95.0 4.5 0.34 5 15.8 52.3 97.7 3.40.27 6 20.7 31.8 95.5 1.6 0.25 7 20.6 40.0 98.6 2.0 0.25 8 19.9 40.798.3 2.1 0.28 9 20.9 40.5 98.1 2.0 0.28 *DPPC/Cholesterol liposomeswhere the DPPC/Chol mole ratio is approximately 1:1. **Only theentrapped amount of amikacin was considered in calculating L/D.

Further information on forming liposomal antiinfective formulations canbe found in PCT/US03/06847, filed Mar. 5, 2003, which is incorporatedherein by reference in its entirety.

Entrapped volume is a basic characteristic of a liposomal formulationand is determined as the volume of intraliposomal aqueous phase per unitof lipid. It is generally expressed in the units of μliters/μmole. Oneoften assumes that when liposomes are formed the concentration of thesolute inside liposomes is equal to that outside in the bulk solution. Ahigher entrapped volume then would lead to higher drug/lipid ratio,i.e., a higher overall drug concentration for the final formulation.

In formulating liposomal amikacin, however, it has been found that theactual drug/lipid ratio that can be produced was more than 3-fold higherthat one would expect based on the entrapped volume. Table 11 shows theresults for 4 different sample preparations of lipid antiinfectiveformulations (see Example 2 in the Exemplification section).

TABLE 11 Amikacin loading into liposomes prepared by different methods.Sample # Measured Parameter 1 2 3 4 Lipids concentration (mg/mL) 35.139.5 50.4 45.0 AMK concentration (mg/mL) 19.9 20.7 10.5 5.0 ActualLipid/Drug (w/w) 1.8 1.9 4.8 9.0 Entrapped volume (ul/umole) 2.4 2.5 2.91.6 Expected Lipid/Drug (w/w) 5.6 6.0 4.1 8.1 Expected/Actual L/D ratio3.19 3.17 0.85 0.90 Liposome Size (um) 0.230 0.217 4.65 3.96Samples 1 and 2 were made by the ethanol infusion procedure disclosedherein, and Samples 3 and 4 were made by liposome formation techniquesknown in the art.

Concentrations of amikacin were measured by immuno-fluorescent assayusing INNOFLUO Seradyn reagent set on TDx analyzer. Lipids were measuredby reverse-phase HPLC using C-8 column and Light scattering detector.

Liposomal volume (volume occupied by liposomes per unit of total volume)in samples #1-3 was determined by measuring the concentration of thefluorescent probe (Sulforhodamine 101 or Carboxyfluorescein) in thetotal volume and in the filtrate volume of the formulation obtained bycentrifugation in CentriSart filtering device. Probe concentration inthe filtrate is higher that the average one due to exclusion of theprobe from the volume occupied by liposomes.

In sample #4, liposomal volume was determined by measuring theconcentration of Potassium ion in a sample after adding fixed amount ofit, 250 ul of KCl (V_(add)) into 10 mL liposomal suspension (V_(o)).Samples were then centrifuged 30 min at 4000 rpm and a supernatant wastaken to measure potassium ion (K) by Cole-Parmer potassium-sensitiveelectrode. Potassium concentration measured was always higher thanexpected due to exclusion of potassium ions from the volume occupied byliposomes. In the control, an equal amount of KCl was added into 10 mLsaline solution. Potassium concentration in control K_(c) was measured.Aqueous and liposomal volumes were than estimated as:

${v_{a} = {{\frac{K_{c}}{K}\left( {V_{o\;} + V_{add}} \right)} - V_{add}}},{v_{L} = {1 - {v_{a}.}}}$

Knowing the liposomal volume and the lipid concentration one candetermine the entrapped volume:

${v_{ent} = \frac{v_{L} - L_{w}}{L_{m}}},$where L_(w) and L_(m) are the weight and molar lipid concentrations,respectively. Lipid density is assumed to be close to 1 mg/mL.Consequently, one can estimate expected Lipid/Drug ratio that the samplewould have if the drug was distributed ideally in the aqueous spacesinside and outside liposomes:

${\left( \frac{L}{D} \right)_{ex} = {\frac{L_{w}}{D_{o}\left( {v_{L} - L_{w}} \right)} = \frac{M_{L}}{D_{o}v_{ent}}}},$where D_(o) is the bulk concentration of the drug during liposomeformation, M_(L) is the average molecular weight of lipids.

As one can see, actual L/D ratios for samples #1 and #2 (1.8 and 1.9)are consistently lower than one would expect from even distribution ofamikacin (5.6 and 6.0), while L/D's for samples #3 and #4 are closer totheoretical values.

A similar comparison was made between 2 sample preparations of a lipidantiinfective formulations where gentamicin sulfate was theantiinfective (see Example 3 in the Exemplification section). The datain Table 12 indicate that the method disclosed herein providesunexpectedly high entrapment of gentamicin. In both samples #5 and #6,the actual Lipid/Drug ratios were almost twice the theoreticallyexpected value.

TABLE 12 Gentamicin loading into liposomes prepared by differentmethods. Sample # Measured Parameter 5 6 Lipids concentration (mg/mL)44.8 41.8 Drug concentration (mg/mL) 14.2 14.9 Actual Lipid/Drug (w/w)3.2 2.8 Entrapped volume (ul/umole) 2.3 2.7 Expected Lipid/Drug (w/w)5.7 5.4 Expected/Actual L/D ratio 1.82 1.92 Liposome Size (um) 0.2260.236

8.4. Drug Release Mediated by P. Aeruginosa Infection

Release of drug in an active form in the vicinity of the infections isan important aspect of the action of liposomal drug formulation of thepresent invention. The potential for such targeted release was tested bymonitoring the release of drug upon incubation with sputum from a CFpatient, release in the lungs of rats pre-inoculated with P. aeruginosa,as well as the activity of against cultures of P. aeruginosa.

The release of amikacin by direct incubation of a culture of P.aeruginosa with a liposomal amikacin formulation of the presentinvention was previously discussed. To further investigate thisphenomenon, a liposomal amikacin formulation was incubated with apreparation of sputum from a cystic fibrosis patient with P. aeruginosainfection. Expectorated sputum was liquefied with bovine DNase I andalginate lyase for 2 hr. at 37° C. A liposomal amikacin formulation orsoluble amikacin (1 mg/mL amikacin) was mixed 1:1: with liquefied sputumor control and incubated at 37° C. with gentle shaking. Aliquots wereanalyzed for amikacin concentration by Abbott TDx Analyzer. Intactliposomes were lysed in a separate aliquot of each sample using adetergent, 1% Triton X-100. Supernatants from each sample were used foranalysis. Over the period of 48 hours, (80-90%) of the amikacin wasreleased in a time-dependent manner from the lipid composition underthese conditions, indicating that drug release may occur at the sites ofinfection in the CF lung.

Release of free drug from liposomes in vivo was compared for rats thathad been instilled with agar beads containing P. aeruginosa (3.5×10⁴CFU/rat) versus those that had not. Three days after bead instillation,rats were allowed to inhale liposomal amikacin formulations of thepresent invention (approx. 6 mg/kg daily dose) every day (no bacteriagroup) or every other day for 14 days (group instilled with beads). 24hours after the last treatment, the total amikacin and free amikacinwere measured as described above. In rats that had received bacteria, anaverage of approximately 50-70% of the detected amikacin was in the freeform, i.e. released from the liposome. In the rats that had not receivedbacteria approximately 20-25% of the drug was in free form. These datastrongly suggest that release of free amikacin from the liposome may bemediated by the presence of P. aeruginosa in vivo.

An in vitro test of release and activity was performed under conditionssimilar to the pharmacokinetics in the lung, where it has beenpreviously shown that free antibiotic is cleared on the time scale of afew hours. Free amikacin or a liposomal amikacin formulation wasincubated with P. aeruginosa PA01 (˜10⁸/mL) in sterile 0.5 mLSlide-A-Lyzer cartridges at varying drug concentrations. Free drugdialyzes out of the cartridges on the time scale of hours under theseconditions. After 24 hrs., the samples were withdrawn from thecartridges and plated to measure CFU. In the preliminary experimentsfree amikacin only slightly reduced the CFU of these samples, while atwo log reduction of CFUs was observed for amikacin comprising lipidcompositions at the same amikacin concentration (50 μg/mL). These datasuggest that amikacin is indeed released in an active form in thepresence of bacteria and that the slow release afforded by theformulation makes more effective use of the drug.

The interaction of the liposomal amikacin formulations of the presentinvention with P. aeruginosa or its virulence factors leads to releaseof amikacin possibly directing release to the site of infection. Whenamikacin is released it is active against P. aeruginosa, and the slowrelease in the vicinity of the bacteria may have an advantage over thenon-specific distribution and rapid clearance of inhaled free drug.

8.5. Effect of Inhaled Liposomal Drug Formations on the Function ofAlveolar Macrophages

The liposomal amikacin formulations of the present invention are in oneembodiment a nanoscale (200-300 nm) liposome-encapsulated form ofamikacin that is formulated to treat chronic P. aeruginosa infections incystic fibrosis patients. It is designed for inhalation with sustainedrelease of amikacin in the lung. Because alveolar macrophages are knownto avidly take up particles in this size range, the effect of theliposomal formulations on these cells is of particular interest. Thebasal and stimulated functions of rat alveolar macrophages obtained bylavage were studied with and without administration of liposomalamikacin formulations and compared to various controls.

Aerosols of the liposomal amikacin formulations, amikacin, placeboliposomes and saline were generated with a PARI LC Star nebulizer andinhaled by CD® IGS female rats in a nose-only inhalation chamber.Inhalation therapy was conducted for 4 hr for 14 consecutive days, suchthat the estimated daily lung dose of total lipid was approximately 12mg/kg for the liposomal amikacin group and 11 mg/kg for the placeboliposome group. Half the rats were euthanized on day 15. The remainingrats were euthanized on day 43. Bronchial alveolar lavage fluid (BALF)was collected from each rat and stored at −80° C. for subsequent assayof nitric oxide (as represented by total nitrates) and tumor necrosisfactor alpha (TNF-α). The cells from the BALF were collected bycentrifugation, counted and cultured in medium with and withoutlipopolysaccharide (LPS) for 24 hr. The supernatants from these cultureswere collected by centrifugation and assayed for nitric oxide and TNF-α.The phagocytic function of BAL macrophages ((10⁶)/mL) was tested bymeasuring the overnight uptake of opsonized fluorescent microspheres(0.2 μm, 2 (10⁹)/mL).

Inhalation of the liposomal amikacin formulation, empty liposomes,soluble amikacin, or saline for 14 consecutive days did not produce asignificant acute or delayed inflammatory response in the lungs of ratsas evident by levels of nitric oxide (nitrates) and TNF-α in BALF whichwere insignificantly different from controls, although there was anearly trend toward higher NO levels in all groups receiving inhalants,including controls. The total recovery of cells was insignificantlydifferent in all groups with an early trend toward morepolymorphonuclear leukocytes in all groups receiving inhalants. Ratalveolar macrophages had normal functions after exposure to the aerosolsof the above test articles despite the fact that they appeared enlargedon day 15 in groups inhaling liposomes. The concentrations of nitratesand TNF-α detected upon culturing of alveolar macrophages in medium onday 15 or 43 of the study were insignificantly different from controls.The macrophages responded normally when stimulated by LPS, producingsubstantial concentrations of nitric oxide (20-40 nmol/10⁶ cells) andTNF-α (5-20 ng/10⁶ cells). These macrophages also had normal phagocyticfunctions, as shown by identical uptake of fluorescent beads compared tountreated controls.

Inhalation of the liposomal amikacin formulations for 14 consecutivedays did not substantially affect the function of alveolar macrophagesin terms of phagocytosis of opsonized beads, production of inflammatorymediators TNF and NO.

9. Dosages

The dosage of any compositions of the present invention will varydepending on the symptoms, age and body weight of the patient, thenature and severity of the disorder to be treated or prevented, theroute of administration, and the form of the subject composition. Any ofthe subject formulations may be administered in a single dose or individed doses. Dosages for the compositions of the present invention maybe readily determined by techniques known to those of skill in the artor as taught herein.

In certain embodiments, the dosage of the subject compounds willgenerally be in the range of about 0.01 ng to about 10 g per kg bodyweight, specifically in the range of about 1 ng to about 0.1 g per kg,and more specifically in the range of about 100 ng to about 50 mg perkg.

An effective dose or amount, and any possible affects on the timing ofadministration of the formulation, may need to be identified for anyparticular composition of the present invention. This may beaccomplished by routine experiment as described herein, using one ormore groups of animals (preferably at least 5 animals per group), or inhuman trials if appropriate. The effectiveness of any subjectcomposition and method of treatment or prevention may be assessed byadministering the composition and assessing the effect of theadministration by measuring one or more applicable indices, andcomparing the post-treatment values of these indices to the values ofthe same indices prior to treatment.

The precise time of administration and amount of any particular subjectcomposition that will yield the most effective treatment in a givenpatient will depend upon the activity, pharmacokinetics, andbioavailability of a subject composition, physiological condition of thepatient (including age, sex, disease type and stage, general physicalcondition, responsiveness to a given dosage and type of medication),route of administration, and the like. The guidelines presented hereinmay be used to optimize the treatment, e.g., determining the optimumtime and/or amount of administration, which will require no more thanroutine experimentation consisting of monitoring the subject andadjusting the dosage and/or timing.

While the subject is being treated, the health of the patient may bemonitored by measuring one or more of the relevant indices atpredetermined times during the treatment period. Treatment, includingcomposition, amounts, times of administration and formulation, may beoptimized according to the results of such monitoring. The patient maybe periodically reevaluated to determine the extent of improvement bymeasuring the same parameters. Adjustments to the amount(s) of subjectcomposition administered and possibly to the time of administration maybe made based on these reevaluations.

Treatment may be initiated with smaller dosages which are less than theoptimum dose of the compound. Thereafter, the dosage may be increased bysmall increments until the optimum therapeutic effect is attained.

The use of the subject compositions may reduce the required dosage forany individual agent contained in the compositions (e.g., theantiinfective) because the onset and duration of effect of the differentagents may be complimentary.

Toxicity and therapeutic efficacy of subject compositions may bedetermined by standard pharmaceutical procedures in cell cultures orexperimental animals, e.g., for determining the LD₅₀ and the ED₅₀.

The data obtained from the cell culture assays and animal studies may beused in formulating a range of dosage for use in humans. The dosage ofany subject composition lies preferably within a range of circulatingconcentrations that include the ED₅₀ with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. For compositions ofthe present invention, the therapeutically effective dose may beestimated initially from cell culture assays.

10. Formulation

The lipid antiinfective formulations of the present invention maycomprise an aqueous dispersion of liposomes. The formulation may containlipid excipients to form the liposomes, and salts/buffers to provide theappropriate osmolarity and pH. The formulation may comprise apharmaceutical excipient. The pharmaceutical excipient may be a liquid,diluent, solvent or encapsulating material, involved in carrying ortransporting any subject composition or component thereof from oneorgan, or portion of the body, to another organ, or portion of the body.Each excipient must be “acceptable” in the sense of being compatiblewith the subject composition and its components and not injurious to thepatient. Suitable excipients include trehalose, raffinose, mannitol,sucrose, leucine, trileucine, and calcium chloride. Examples of othersuitable excipients include (1) sugars, such as lactose, and glucose;(2) starches, such as corn starch and potato starch; (3) cellulose, andits derivatives, such as sodium carboxymethyl cellulose, ethyl celluloseand cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin;(7) talc; (8) excipients, such as cocoa butter and suppository waxes;(9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil,olive oil, corn oil and soybean oil; (10) glycols, such as propyleneglycol; (11) polyols, such as glycerin, sorbitol, and polyethyleneglycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar;(14) buffering agents, such as magnesium hydroxide and aluminumhydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonicsaline; (18) Ringer's solution; (19) ethyl alcohol; (20) phosphatebuffer solutions; and (21) other non-toxic compatible substancesemployed in pharmaceutical formulations.

EXEMPLIFICATION Example 1

The following is a detailed description of the manufacture of 150 mL ofLiposomal/complexed amikacin.

Total Intial Volume=1.5 L

Ethanol Content=23.5% (v/v)

Lipid Composition: DPPC/Chol (1:1 mole ratio)

Intial [Lipid]=7.6 mg/mL

Intial [amikacin sulfate]=57.3 mg/mL

Final product Volume=150 mL

I) Compounding and Infusion:

7.47 g DPPC and 3.93 g Cholesterol were dissolved directly in 352.5 mLethanol in a 50 C water bath. 85.95 g amikacin sulfate was dissolveddirectly in 1147.5 mL PBS buffer. The solution is then titrated with IONNaOH or KOH to bring the pH to approximately 6.8.

352.5 mL ethanol/lipid was added or infused to the 1147.5 mLamikacin/buffer to give a total initial volume of 1.5 L. Theethanol/lipid was pumped @−30 mL/min (also called infusion rate) with aperistaltic pump into the amikacin/buffer solution which was beingrapidly stirred at 150 RPM in a reaction vessel on a stir plate at roomtemperature

The product was stirred at room temperature for 20-30 minutes.

II) Diafiltration or “Washing” Step:

The mixing vessel was hooked up to a peristaltic pump and diafiltrationcartridge. The diafiltration cartridge is a hollow membrane fiber with amolecular weight cut-off of 500 kilodaltons. The product was pumped fromthe reaction vessel through the diafiltration cartridge and then backinto the mixing vessel at room temperature. A back pressure ofapproximately 7 psi is created throughout the cartridge. Free amikacinand ethanol was forced through the hollow fiber membrane by the backpressure leaving the liposomal amikacin (product) behind. The productwas washed 8 times at room temperature. Fresh PBS buffer was added (viaanother peristaltic pump) to the reaction vessel to compensate for thepermeate removal and to keep a constant product volume.

The product was concentrated.

Example 2

High Liposomal Entrapment of Amikacin.

Four samples of lipid antiinfective formulations were prepared atvarious lipid and antiinfective concentrations according to thefollowing procedures.

Sample #1. Amikacin sulfate 1.72 kg was dissolved in 23 Liters salinesolution (0.9% NaCl) and pH was adjusted to 6.5 by adding necessaryamount NaOH. Lipids—98.2 g DPPC and 51.8 g Cholesterol were dissolved in7 liters ethanol. Liposomes were formed by infusion of lipid solutioninto amikacin solution at a rate of ˜600 mL/min and under constantstirring. Resulting suspension was then washed to remove ethanol andun-entrapped amikacin by diafiltration using an Amersham Hollow Fibercartridge 500 kD pore size. The suspension was concentrated to a finalvolume of ˜3.5 L.Sample #2. The procedure was similar to that for sample #1 with allmaterial quantities scaled down 100 fold. Amikacin sulfate 172 g wasdissolved in 230 mL saline solution (0.9% NaCl) and pH was adjusted to6.6 by adding necessary amount NaOH. Lipids—0.982 g DPPC and 0.518 gCholesterol were dissolved in 70 mL ethanol. Liposomes were formed byinfusion of the lipid solution into the amikacin solution at a rate of˜300 mL/min and under constant stirring. The resulting suspension wasthen washed to remove ethanol and un-entrapped amikacin by diafiltrationusing an Amersham Hollow Fiber cartridge. The suspension wasconcentrated to a final volume of ˜35 mL.Sample #3. Liposomes were made by a procedure known as SPLV. Amikacinsulfate 1.4 g was dissolved in 20 mL saline solution (0.9% NaCl) makingpH 3.3. Lipids, 0.666 g DPPC and 0.333 g Cholesterol were dissolved in40 mL dichloromethane. Amikacin and lipid solutions were mixed togetherin a 500 mL round flask and briefly sonicated to form an emulsion. Flaskwas then connected to a BUCHI Rotavapor system to remove dichloromethaneat low vacuum (−5 inches Hg) and temperature 50° C. and constantrotation until the amikacin-lipid mixture formed a gel. When the geleventually collapsed, vacuum was gradually increased to −20 inches Hgand drying continued for 30 more minutes. The final volume of formedliposomal suspension was 22 mL.Sample #4. The procedure was similar to that for sample #3. Amikacinsulfate 1.3 g was dissolved in 20 mL of saline solution, and pH wasadjusted to 6.5 by adding NaOH. Lipids, 0.583 g DPPC and 0.291 gCholesterol were dissolved in 35 mL dichloromethane. The sonication stepwas skipped. The solvent removal step on the Rotavapor system wascarried out at 40° C. for 2 hr. Final volume was 20 mL.

Example 3

High Liposomal Entrapment of Gentamicin

Sample #5. Gentamicin sulfate 20.0 g was dissolved in 230 mL salinesolution (0.9% NaCl) and pH was adjusted to 6.5 by adding necessaryamount of sulfuric acid. Lipids—0.982 g DPPC and 0.518 g Cholesterolwere dissolved in 70 mL ethanol, Liposomes were formed by infusion oflipid solution into gentamicin solution at a rate of ˜500 nil/min andunder constant stirring. Un-entrapped gentamicin and ethanol wereremoved by diafiltration using an Amersham Hollow Fiber cartridge. Thesuspension was concentrated to a final volume of ˜35 mL.Sample #6. The procedure was similar to that for sample #5, except:Gentamicin sulfate 17.0 g was dissolved in 230 mL Na₂SO₄ 100 mM solutionand pH was adjusted to 6.5 by adding necessary amount of H₂SO₄.Lipids—0.982 g DPPC and 0.518 g Cholesterol were dissolved in 75 nitethanol.

Example 4

Entrapment of Other Salt Forms of Amikaein

Sample #7. The procedure was similar to that for sample #2 under Example2. Amikacin base 10.7 g and Citric acid 4.2 g were dissolved in 230 mLsaline solution (0.9% NaCl). pH of resulted amikacin-citrate solutionwas 6.2. Lipids—0.982 g DPPC and 0.518 g Cholesterol were dissolved in70 mL ethanol. Liposomes were formed by infusion of lipid solution intoamikacin solution at a rate of ˜500 mL/min and under constant stirring.Un-entrapped amikacin and ethanol were removed by diafiltration using anAmersham Hollow Fiber cartridge. The suspension was concentrated to afinal volume of ˜35 mL.

The actual Lipid/Drug ratio was similar to that for sample #2 and againlower than expected (Drug entrapment higher than expected). Consideringthe fact that the entrapped volume in the sample #7 was only 1.5(compared to 2.5 for sample #2), the Expected/Actual L/D ratio was ashigh as 5.2. Thus, liposomal amikacin citrate, like amikacin sulfate,can also be formulated with high entrapment.

TABLE 13 Samples 5-7 parameter summary. Sample # Measured Parameter 5 67 Lipids concentration (mg/mL) 44.8 41.8 41.7 Drug concentration (mg/mL)14.2 14.9 17.8 Actual Lipid/Drug (w/w) 3.2 2.8 2.3 Entrapped volume(ul/umole) 2.3 2.7 1.5 Expected Lipid/Drug (w/w) 5.7 5.4 12.2Expected/Actual L/D ratio 1.82 1.92 5.20 Liposome Size (um) 0.226 0.2360.234

Example 5

Bioavailability of Amikacin from Inhaled Liposomal Amikacin Formulationsin the Rat

The rate of release of amikacin from the liposomes was measured afterinhalation by rats and compared to inhaled soluble amikacin.

The test items were aerosolized via a Pari LC Star nebulizer attached toa nose-only inhalation chamber. CD® IGS rats received an estimated lungdeposited dose of 6 mg/kg of amikacin in the form of a liposomalformulation or 5 mg/kg of soluble amikacin as a single dose or doseddaily for 14 consecutive days. Lung or other tissue was homogenized witha Polytron apparatus. The kinetics of clearance of amikacin from thelung was examined by analysis of lung homogenates at varying time pointsafter the single dose treatment or 1 day and 28 days afteradministration of the multiple doses. Amikacin levels were measured byimmunofluorescence polarization on a Abbott TDx® analyzer in the absenceor presence of 1% Triton X-100, which releases amikacin from liposomes.Whole lung samples were spiked with liposomes before homogenization totest the release of amikacin under these conditions. Free and totalamikacin were measure with and without 1% Triton X-100 to assess leakageof drug.

Liposomal amikacin, spiked into whole lung samples, showed nosignificant release of amikacin as a result of tissue homogenizationwith the Polytron homogenizer in the absence of this detergent. However,addition of 1% Triton X-100 led to recovery of all of the expected drug.Therefore a direct comparison could be made of the total level ofamikacin (with detergent) versus the freely available levels in lungtissue.

A high total concentration of amikacin (approx. 500-600 μg/g of lungtissue) was observed immediately after the 6 mg/kg single dose of theliposomal amikacin, which slowly decreased by about 50% over a 7 dayperiod. The temporal profile for the release of free amikacin from theseliposomes showed an initial high concentration of free drug, probablyresulting from amikacin liberated as a result of nebulization. Thisphase was followed by a nadir at about 24 hours and a subsequentincrease, reaching a maximum of 279 μg/g at 96 hours afteradministration. By the end of the 7 day experiment, a substantialportion of drug remaining in the lung was in the free form(approximately 50-70%). It appeared that a small portion of the solubledrug administered by inhalation also remained for a long period of timein the lung. However, most of the amikacin administered in soluble formwas cleared within several hours, and the apparent free amikacin AUCover 7 days was at least 2× higher for the liposomal amikacin animalsthan for those that received soluble amikacin. Some aspects of thisbehavior can be qualitatively modeled with appropriate rate constantsfor clearance and slow release of drug from liposomes.

After 14 consecutive days of administration (24 hours after the lastdose), more than 20% of the total amikacin in the lungs of rats thatreceived liposomal amikacin was present as free drug (approximately 650μg/g). The total free drug level was even greater than the amount inrats that inhaled soluble amikacin (approx. 500 μg/g).

Free amikacin is released slowly from the liposomes of the liposomalamikacin formulations in the lungs of healthy animals over a time scaleof days. The free drug that is released has a relatively long residencetime in the lung as seen by a substantial depot of free drug in thelungs.

Example 6

In-Line Infusion Process

The essence of the In-Line infusion process is that a stream of lipidsolution is mixed with a stream of antiinfective solution “in-line” via,for example, a Y-connector which connects to a length of tubing, termeda mixing tube, where further mixing can occur. In this regard, this newprocess differs from the ‘conventional’ ethanol infusion process, wherelipid solution is infused as a stream into a bulk of amikacin solution.

Amikacin and Lipid Solutions Preparation.

Amikacin sulfate 12.0 g was dissolved in 200 mL water and pH wasadjusted to 6.5 by adding necessary amounts of 25% NaOH solution.Lipids, 1.480 g DPPC and 0.520 g cholesterol, were dissolved in amixture of 60 mL ethanol and 10 mL water. These amounts result in a 300mL batch after infusion at a lipid/amikacin flow rate of 300/500 mL/min,respectively. Volumes can be proportionally adjusted for larger scale orif different flow rates are desired.

The amikacin solution prepared according to above results inapproximately 40 mg/mL amikacin (per base) solution. The lipid solutionas presented was DPPC/Chol (mole ratio of 60/40) with a total lipid ofapproximately 20 mg/mL solution (90% ethanol). Lipids were heated to˜40° C. for faster dissolution.

The exact amounts needed for a 300 mL batch are: amikacin 150 mL, lipid90 mL, and 60 mL of additional saline (or water) which is added after orduring infusion to adjust final ethanol concentration.

Manufacturing Procedure.

One embodiment of the infusion system is shown in FIG. 8.

Lipid and Amikacin solutions are mixed in-line using a Y-connector (ID3.2 mm, OD 6.4 mm) at flow rates ˜300/500 mL/min (i.e. ˜1/1.67 volumeratio instead of ˜1/3.35 in the conventional process). A MasterFlex tubeL/S 25 (ID 4.8 mm) was used to deliver the lipid solution and a L/S 17tube (ID 6.4 mm) was used to deliver the amikacin solution. To obtainsynchronous flow rates, two pump heads with one MasterFlex drive wereused. According to the tube cross-section areas, the theoretical flowrate ratio should be 4.8²/6.4²=0.562=1/1.78. When the pump drive was setto 500 mL/min for Amikacin tube L/S 17, the measured flow rates were˜300/500=1/1.67.

Since the lipid solution contains 90% ethanol, the in-line mixture had˜34% ethanol. To prevent amikacin precipitation, NaCl solution can beadded after or during infusion at a flow rate 100-200 mL/min (it isassumed that the liposomes are already formed at this point). Thus thefinal mixture would have ˜27% ethanol, of which all free amikacin isexpected to be soluble.

Total liquid infusion flow rate, 800-1000 mL/min, is comparable to thepermeate flow rate when using two big diafiltration cartridges. Thismakes it possible to do simultaneous infusion and concentration bydiafiltration.

The resulting liposome suspension was washed to remove free amikacin bydiafiltration using an Amersham hollow fiber cartridge UFP-500-C-3MA(membrane area 140 cm², fiber ID 0.5 mm). In the first step, thesuspension was concentrated to nearly half of the original volume (150mL). Then, during diafiltration to wash, the suspension wasre-circulated and fresh saline solution was fed into the mixture at arate of ˜6 mL/min in order to match the permeate rate and thus maintaina constant volume. Diafiltration continued until 4 times the suspensionvolume of the feeding saline solution was dispensed (i.e., 4*150 mL=600mL). This diafiltration/washing procedure will be referred to as 4“washes”. Finally, the suspension was concentrated (diafiltrationwithout saline input) to obtain the Final Product at a desired amikacinand lipid concentration. The recirculation flow rate during thediafiltration step was ˜350 mL/min, and during the final concentrationstep it was gradually reduced to ˜150 mL/min in order to maintain theinlet pressure below 10 PSI.

REFERENCES

-   1. Veldhuizen, R., Nag, K., Orgeig, S, and Possmayer, F., The Role    of Lipids in Pulmonary Surfactant, Biochim. Biophys. Acta    1408:90-108 (1998).-   2. Hagwood, S., Derrick, M. and Poulain, F., Structure and    Properties of Surfactant Protein B, Biochim. Biophys. Acta    1408:150-160 (1998).-   3. Johansson, J., Structure and Properties of Surfactant ProteinC,    Biochim. Biophys. Acta 1408:161472 (1998).-   4. Ikegami, M. and Jobe, A. H., Surfactant Protein Metabolism in    vivo, Biochim. Biophys. Acta 1408:218-225 (1998).-   5. Couveur, P., Fattel, E. and Andremont, A., Liposomes and    Nanoparticles in the Treatment of Intracellular Bacterial    Infections, Pharm. Res. 8:1079-1085 (1991).-   6, Gonzales-Rothi, R. J., Casace, J., Straub, L., and Schreier, H.,    Liposomes and Pulmonary Alveolar Macrophages: Functional and    Morphologic Interactions, Exp. Lung Res. 17:685-705 (1991).-   7. Swenson, C. E., Pilkiewicz, F. G., and Cynamon, M. H., Liposomal    Aminoglycosides and TLC-65 Aids Patient Care 290-296 (December,    1991).-   8. Costerton, J. W., Stewart, P. S., and Greenberg, E. P., Bacterial    Biofilms: A Common Cause of Persistent Infections, Science    284:1318-1322 (1999).-   9. Cash, H. A., Woods, D. E., McCullough, W. G., Johanson, J. R.,    and Bass, J. A., A Rat Model of Chronic Respiratory Infection with    Pseudomonas aeruginosa, American Review of Respiratory Disease    119:453-459 (1979).-   10. Cantin, A. M. and Woods, D. E. Aerosolized Prolastin Suppresses    Bacterial Proliferation in a Model of Chronic Pseudomonas aeruginosa    Lung Infection, Am. J. Respir. Crit, Care Med. 160:1130-1135 (1999).-   11. Ramsey, B. W., Dorkin, H. L., Eisenberg, J. D., Gibson, R. L.,    Harwood, I. R., Kravitz, R. M., Efficacy of Aerosolized Tobramycin    in Patients with cystic Fibrosis. New England J. of Med.    328:1740-1746 (1993).-   12. Mendelman, P. M., Smith, A. L., Levy, J., Weber, A., Ramsey, B.,    Davis, R. L., Aminoglycoside Penetration, Inactivation, and Efficacy    in Cystic Fibrosis Sputum, American Review of Respiratory Disease    132:761-765 (1985).-   13. Price, K. E., DeFuria, M. D., Pursiano, T. A. Amikacin, an    aminoglycoside with marked activity against antibiotic-resistant    clinical isolates. J Infect Dis 134:S249261 (1976).-   14. Darnaso, D., Moreno-Lopez, M., Martinez-Beltran, J.,    Garcia-Iglesias, M. C. Susceptibility of current clinical isolates    of Pseudomonas aeruginosa and enteric gram-negative bacilli to    Amikacin and other aminoglycoside antibiotics. J Infect Dis    134:5394-90 (1976).-   15. Pile, J. C., Malone, J. D., Eitzen, E. M., Friedlander, A. M.,    Anthrax as a potential biological warfare agent. Arch. Intern. Med.    158:429-434 (1998).-   16. Gleiser, C. A., Berdjis, C. C., Hartman, H. A., &    Glouchenour, W. S., Pathology of experimental respiratory anthrax in    Macaca mulatta. Brit, J. Exp, Path., 44:416-426 (1968).

INCORPORATION BY REFERENCE

Publications and references, including but not limited to patents andpatent applications, cited in this specification are herein incorporatedby reference in their entirety in the entire portion cited as if eachindividual publication or reference were specifically and individuallyindicated to be incorporated by reference herein as being fully setforth. Any patent application to which this application claims priorityis also incorporated by reference herein in the manner described abovefor publications and references.

EQUIVALENTS

While this invention has been described with an emphasis upon preferredembodiments, it will be obvious to those of ordinary skill in the artthat variations in the preferred devices and methods may be used andthat it is intended that the invention may be practiced otherwise thanas specifically described herein. Accordingly, this invention includesall modifications encompassed within the spirit and scope of theinvention as defined by the claims that follow.

What is claimed:
 1. A liposomal aminoglycoside formulation comprising aliposome having a lipid bilayer and an aminoglycoside or apharmaceutically acceptable salt thereof encapsulated therein, whereinthe lipid bilayer comprises a neutral phospholipid and a sterol, theweight ratio of lipid to the aminoglycoside, or the pharmaceuticallyacceptable salt thereof in the formulation is 0.75:1 or less and theliposome has a mean diameter of about 0.1 μm to about 1.0 μm.
 2. Theliposomal aminoglycoside formulation of claim 1, wherein theaminoglycoside is amikacin, gentamicin, tobramycin, streptomycin,netilmicin, kanamycin, or a pharmaceutically acceptable salt thereof. 3.The liposomal aminoglycoside formulation of claim 1, wherein theaminoglycoside is amikacin, or a pharmaceutically acceptable saltthereof.
 4. The liposomal aminoglycoside formulation of claim 1, whereinthe aminoglycoside is streptomycin, or a pharmaceutically acceptablesalt thereof.
 5. The liposomal aminoglycoside formulation of claim 1,wherein the aminoglycoside is tobramycin, or a pharmaceuticallyacceptable salt thereof.
 6. The liposomal aminoglycoside formulation ofclaim 1, wherein the neutral phospholipid is a phosphatidylcholine. 7.The liposomal aminoglycoside formulation of claim 1, wherein the neutralphospholipid is dipalmitoylphosphatidylcholine (DPPC).
 8. The liposomalaminoglycoside formulation of claim 1, wherein the sterol ischolesterol.
 9. The liposomal aminoglycoside formulation of claim 1,wherein the neutral phospholipid is DPPC, and the sterol is cholesterol.10. The liposomal aminoglycoside formulation of claim 1, wherein theaminoglycoside is amikacin or a pharmaceutically acceptable saltthereof, the neutral phospholipid is DPPC and the sterol is cholesterol.11. The liposomal aminoglycoside formulation of claim 1, wherein theaminoglycoside is an aminoglycoside sulfate.
 12. The liposomalaminoglycoside formulation of claim 1, wherein the aminoglycoside isamikacin sulfate.
 13. The liposomal aminoglycoside formulation of claim1, wherein the aminoglycoside is tobramycin sulfate.
 14. The liposomalaminoglycoside formulation of claim 1, wherein the aminoglycoside isstreptomycin sulfate.
 15. The liposomal aminoglycoside formulation ofclaim 1, wherein the liposome has a mean diameter of about 0.2 μm toabout 1.0 μm.
 16. The liposomal aminoglycoside formulation of claim 1,wherein the liposome has a mean diameter of about 0.2 μm to about 0.5μm.
 17. A nebulized spray comprising the liposomal aminoglycosideformulation of claim
 1. 18. A nebulized spray comprising the liposomalaminoglycoside formulation of claim
 3. 19. A nebulized spray comprisingthe liposomal aminoglycoside formulation of claim
 6. 20. A nebulizedspray comprising the liposomal aminoglycoside formulation of claim 7.21. A nebulized spray comprising the liposomal aminoglycosideformulation of claim
 8. 22. A nebulized spray comprising the liposomalaminoglycoside formulation of claim
 9. 23. A nebulized spray comprisingthe liposomal aminoglycoside formulation of claim
 10. 24. A method oftreating a patient for a pulmonary infection comprising administering tothe patient a therapeutically effective amount of the liposomalaminoglycoside formulation of claim
 1. 25. The method of claim 24,wherein the pulmonary infection is a Pseudomonas, staphylococcal,streptococcal, Escherichia, Kiebsiella, Enterobacter, Serratia,Haemophilus, Yersinia, Burkholderia, or mycobacterial infection.
 26. Themethod of claim 24, wherein the pulmonary infection is a mycobacterialinfection.
 27. The method of claim 24, wherein the pulmonary infectionis a Pseudomonas infection.
 28. The method of claim 24, wherein theliposome has a mean diameter of about 0.2 μm to about 1.0 μm.
 29. Themethod of claim 24, wherein the liposome has a mean diameter of about0.2 μm to about 0.5 μm.
 30. The method of claim 24, wherein the patientis a cystic fibrosis patient.
 31. The method of claim 24, wherein theaminoglycoside is amikacin or a pharmaceutically acceptable saltthereof.
 32. The method of claim 25, wherein the mycobacterial infectionis Mycobacterium tuberculosis, Mycobacterium avium complex (MAC)(Mycobacterium avium and Mycobacterium intracellulare), Mycobacteriumkansasii, Mycobacterium xenopi, Mycobacterium marinum, Mycobacteriumulcerans, or Mycobacterium fortuitum complex (M. fortuitum and M.chelonae).
 33. The method of claim 27, wherein the Pseudomonas infectionis a Pseudomonas aeruginosa infection.
 34. The method of claim 27,wherein the Pseudomonas infection is a chronic Pseudomonas infection.35. The method of claim 32, wherein the mycobacterial infection is aMycobacterium avium complex (MAC) (Mycobacterium avium and Mycobacteriumintracellulare) infection.
 36. The method of claim 34, wherein thechronic Pseudomonas infection is a chronic Pseudomonas aeruginosainfection.
 37. The method of claim 26, wherein the aminoglycoside isamikacin sulfate.
 38. The method of claim 27, wherein the aminoglycosideis amikacin sulfate.
 39. The method of claim 28, wherein theaminoglycoside is amikacin sulfate.
 40. The method of claim 29, whereinthe aminoglycoside is amikacin sulfate.
 41. The method of claim 30,wherein the aminoglycoside is amikacin sulfate.
 42. The method of claim31, wherein the aminoglycoside is amikacin sulfate.
 43. The method ofclaim 32, wherein the aminoglycoside is amikacin sulfate.
 44. The methodof claim 33, wherein the aminoglycoside is amikacin sulfate.
 45. Themethod of claim 34, wherein the aminoglycoside is amikacin sulfate. 46.The method of claim 35, wherein the aminoglycoside is amikacin sulfate.47. The method of claim 36, wherein the aminoglycoside is amikacinsulfate.
 48. The method of claim 26, wherein the aminoglycoside isamikacin sulfate.
 49. The method of claim 37, wherein the neutralphospholipid is DPPC and the sterol is cholesterol.
 50. The method ofclaim 38, wherein the neutral phospholipid is DPPC and the sterol ischolesterol.
 51. The method of claim 39, wherein the neutralphospholipid is DPPC and the sterol is cholesterol.
 52. The method ofclaim 40, wherein the neutral phospholipid is DPPC and the sterol ischolesterol.
 53. The method of claim 41, wherein the neutralphospholipid is DPPC and the sterol is cholesterol.
 54. The method ofclaim 42, wherein the neutral phospholipid is DPPC and the sterol ischolesterol.
 55. The method of claim 43, wherein the neutralphospholipid is DPPC and the sterol is cholesterol.
 56. The method ofclaim 44, wherein the neutral phospholipid is DPPC and the sterol ischolesterol.
 57. The method of claim 45, wherein the neutralphospholipid is DPPC and the sterol is cholesterol.
 58. The method ofclaim 46, wherein the neutral phospholipid is DPPC and the sterol ischolesterol.
 59. A nebulized spray comprising the liposomalaminoglycoside formulation of claim
 11. 60. A nebulized spray comprisingthe liposomal aminoglycoside formulation of claim 12.